Fluorescent Metal Ion Indicators with Large Stokes Shifts

ABSTRACT

The present invention provides fluorogenic compounds for the detection of target metal ions wherein the compounds exhibit a Stokes shift greater than 50 nm and the detectable signal is modulated by photoinduced electron transfer (PET). The present compounds consist of three functional elements, the ion sensing moiety (chelating moiety), the reporter moiety (fluorophore or fluorescent protein) and spacer or linker between the sensing and reporter moieties of the present compound that allows for PET upon binding of a metal ion and excitation by an appropriate wavelength.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/651,138, filedDec. 31, 2009, which is a continuation of U.S. Ser. No. 12/484,903, nowabandoned, filed Jun. 15, 2009, which is a continuation of U.S. Ser. No.12/180,273, now abandoned, filed Jul. 25, 2008, which is a continuationof U.S. Ser. No. 11/191,799, now abandoned, filed Jul. 27, 2005, whichclaims priority to U.S. Ser. No. 60/591,750, filed Jul. 27, 2004, whichdisclosures are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to novel compositions and methods for thedetection and isolation of metal ions, including physiologicalconcentrations of calcium. The invention has applications in the fieldsof cell biology, neurology, immunology and proteomics.

BACKGROUND OF THE INVENTION

Metal ions play an important role in biological systems. Cells utilizemetal ions for a wide variety of functions, such as regulating enzymeactivity, protein structure, cellular signaling, as catalysts, astemplates for polymer formation and as regulatory elements for genetranscription. Metal ions can also have a deleterious effect whenpresent in excess of bodily requirements or capacity to excrete. A largenumber of natural and synthetic materials are known to selectively ornon-selectively bind to or chelate metal ions. Ion chelators arecommonly used in solution for in vivo control of ionic concentrationsand detoxification of excess metals, and as in vitro buffers. When boundto a fluorophore, ion chelators are typically used as optical indicatorsof ions and are useful in the analysis of cellular microenvironments ordynamic properties of proteins, membranes and nucleic acids.

Such indicators are also useful for measuring ions in extracellularspaces; in vesicles; in vascular tissue of plants and animals;biological fluids such as blood and urine; in fermentation media; inenvironmental samples such as water, soil, waste water and seawater; andin chemical reactors. Optical indicators for ions are important forqualitative and quantitative determination of ions, particularly inliving cells. Fluorescent indicators for metal cations also permit thecontinuous or intermittent optical determination of these ions in livingcells, and in solutions containing the ions.

A variety of fluorescent indicators that are useful for the detection ofbiologically relevant soluble free metal ions (such as Ca²⁺, Mg⁺ andZn²⁺) have been described that utilize oxygen-containing anionic orpolyanionic chelators to bind to metal ions. In particular, fluorescentindicators utilizing a polycarboxylate BAPTA chelator have beenpreviously described (U.S. Pat. No. 4,603,209 to Tsien et al. (1986);U.S. Pat. No. 5,049,673 to Tsien et al. (1991); U.S. Pat. No. 4,849,362to DeMarinis et al. (1989); U.S. Pat. No. 5,453,517 to Kuhn et al.(1995); U.S. Pat. No. 5,501,980 to Malekzadeh et al. (1996); U.S. Pat.No. 5,459,276 to Kuhn et al. (1995); U.S. Pat. No. 5,501,980 toKaterinopoulos et al. (1996); U.S. Pat. No. 5,459,276 to Kuhn et al.(1995).

In general, a useful property for metal ion indicators is the ability todetect and/or quantify a selected metal ion in the presence of othermetal ions. Discrimination of Ca²⁺, Na⁺ and K⁺ ions in the presence ofother metal ions is particularly useful for certain biological orenvironmental samples. For most biological applications, it is essentialthat the indicators be effective in aqueous solutions. It is also usefulthat indicators for biological applications be relatively insensitive topH changes over the physiological range (pH 6-8) and sensitive to ionconcentrations in the physiological range (for calcium, a K_(d) of about100 μM to about 100 nM). It is also beneficial if the indicator absorbsand emits light in the visible spectrum where biological materials havelow intrinsic absorbance or fluorescence.

Also useful are chelators that possess a chemically reactive functionalgroup, so that the chelating group can be attached to polymers for usein remote sensing of ions or enhancing the solubility or localization ofthe optical sensor. Many chelators bind to intracellular proteins,altering the chelator's metal binding properties. In addition, due totheir relatively small size, they are readily sequesterednon-selectively in intracellular vesicles, further limiting theireffectiveness. One means of circumventing these problems is to attachthe chelate compound to a large, water-soluble polysaccharide, such asdextran or FICOL, by means of modification of the polysaccharide toallow covalent attachment of the indicator. Dextrans and FICOLs areespecially suitable for this application, as they are low cost,optically transparent above about 250 nm and available in multipleranges of molecular weights. Furthermore, polysaccharides and theirconjugates are reasonably compatible with most biological materials anddo not interact significantly with intracellular components. Althoughfluorescent polysaccharides have been previously described, as haveindicator conjugates of dextrans, none possess the advantageousproperties of the indicator conjugates of the current invention.

The chelators of the invention show significant ability to discriminatebetween metal ions under physiological conditions, particularly Ca²⁺,Na⁺ and K⁺ ions. This selectivity can be tailored by careful selectionof chelate substituents. The compounds of the invention are typicallysoluble in aqueous solutions.

The compounds of the invention that act as indicators for target ionsabsorb and emit light in the visible spectrum and possess significantutility as a means of detecting and quantifying certain metal ion levelsin living cells, biological fluids or aqueous solutions. Upon bindingthe target ion in the chelating moiety of the indicator, the opticalproperties of the attached fluorophore are generally affected in adetectable way by photoinduced electron transfer (PET), and this changeis correlated with the presence of the ion according to a definedstandard. Compounds having relatively long wavelength excitation andemission bands can be used with a variety of optical devices and requireno specialized (quartz) optics, such as are required by indicators thatare excited or that emit at shorter wavelengths. These indicators aresuitable for use in fluorescence microscopy, flow cytometry,fluoroscopy, or any other application that currently utilize fluorescentmetal ion indicators.

The distinguishing feature of the present compounds is the alkylenespacer between the chelating moiety and the reporter moiety, thuslimiting the interaction between the ion sensor and reporter to the PETmechanism. Although several metal sensors based on PET are known in theart (U.S. Pat. Nos. 6,124,135; 6,359,135; He et al. Chem. Soc. (2003)125:1468-1469; He et al. Anal. Chem. (2003) 75:3549-55), they arelimited to the non-charged crown ether or cryptand moieties, which areunable to interact with calcium ions, an important physiological metalion. Also the reported PET sensors employ a different linkage (formed byalkylation reaction, rather than acylation utilized in this invention)between the functional elements. The present compounds provide a highaffinity for calcium ions and a larger Stokes shift compared to otherBAPTA-based calcium indicators (U.S. Pat. No. 5,049,673). The presentinvention provides an improvement over known calcium indicators, whichhas many important implications including the use in multicolorfluorescent assays.

SUMMARY OF THE INVENTION

The present invention provides a novel class of fluorogenic metal ionindicators that produce a detectable signal that is modulated byphotoinduced electron transfer (PET), compositions, methods of use andkits for detecting metal ions in a sample. The metal ions bound anddetected by the present compounds include, but are not limited to, Ca²⁺,Zn²⁺, Mg²⁺, Ga³⁺, Tb³⁺, La³⁺, Pb²⁺, Hg²⁺, Cd²⁺, Cu²⁺, Ni²⁺, Co²⁺, Fe²⁺,Mn²⁺, Ba²⁺, and Sr²⁺. Particularly relevant in biological systems arethe metal ions selected from the group consisting of Ca²⁺, Mg²⁺, Fe²⁺and Zn²⁺.

The present compounds exhibit a Stokes shift great than about 50 nm,preferably greater than about 100 nm, more preferably greater than 150nm and most preferably greater than 200 nm when bound by a metal ioncapable of being chelated by the chelating moiety and illuminated withan appropriate wavelength. In certain aspects, the present compoundsexhibit a Stokes shift greater than about 250 nm.

In an exemplary embodiment, a present compound for the detection ofmetal ions wherein a detectable response is a result of photoinducedelectron transfer (PET), comprise a metal chelating moiety and afluorophore or a fluorescent protein (reporter moiety) that iscovalently bonded to the metal chelating moiety by linker —(CR₂)_(n)NR′—or —(CR₂)_(n)— wherein R and R′ are independently selected from thegroup consisting of hydrogen, alkyl, and substituted alkyl and n is1-10. When the linker is —(CR₂)_(n)— a terminal carbon must be directlyand covalently bonded to a nitrogen atom of the fluorophore. In afurther embodiment the present compound is utilized to bind and detectcalcium ions wherein a detectable response is a result of photoinducedelectron transfer (PET), wherein the compound comprises a metalchelating moiety that is capable of binding calcium ions and afluorophore that is covalently bonded to the metal chelating moiety by alinker —(CR₂)_(n)NR′— or —(CR₂)_(n)— wherein R and R′ are independentlyselected from the group consisting of hydrogen, alkyl, and substitutedalkyl and n is 1-10. Again, if the linker is —(CR₂)_(n)— a terminalcarbon of the linker must be bound to a nitrogen atom of thefluorophore.

Many chelating moieties are known that bind metal ions and find use indetecting metal ions in biological systems. BAPTA is one such chelatingmoiety that is well known for its ability to bind and detect calciumions when conjugated to a reporter molecule. Thus, the use of the BAPTAmoiety in conjunction with the present linkers and reporter moietyprovides a novel compound that represents an improvement over knowncalcium indicators. These present calcium indicators demonstrateimproved affinity for calcium ions and a larger Stokes shift incomparison to other BAPTA-based calcium indicators such as the indicatorsold under the trade name Fluo-3 (Molecular Probes, Inc.)

A present compound based on the BAPTA chelator has the formula:

wherein R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are independently H or C₁-C₆ alkyl; andR¹³ and R¹⁴ are independently hydrogen, C₁-C₆ alkyl, —CH₂OCOCH₃ or asalt ion;R¹-R⁸ are selected independently from the group consisting of hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl, heteroaryl, amino(—NR¹⁹R²⁰), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether,sulfonyl, reactive group, carrier molecule, solid support, reportermolecule, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore and—(CR₂)_(n)-fluorophore or a member selected from R¹ in combination withR²; R² in combination with R³; R³ in combination with R⁴; R⁵ incombination with R⁶; R⁶ in combination with R⁷; and R⁷ in combinationwith R⁸ together with the atoms to which they are joined, form a ringwhich is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, asubstituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;wherein R, and R′ are independently selected from the group consistingof hydrogen, alkyl, and substituted alkyl;R″ is independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, fluorophore, carrier molecule, solid supportand reactive group; and n is 1-10; andwherein R¹⁹ and R²⁰ are independently selected from the group consistingof H, C₁-C₆ alkyl, substituted alkyl, C₁-C₆ carboxyalkyl(—(CH₂)₁₋₆COOR¹³), an alpha-acyloxyalkyl, a biologically compatiblesalt, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl,heteroaryl, and substituted heteroaryl;R⁹, R¹⁰, R¹¹ and R¹², are independently selected from the groupconsisting of hydrogen, a reactive group, a carrier molecule, a solidsupport, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore and C₁-C₆ alkyl, ora member selected from R⁹ in combination with R¹⁰; or R¹¹ in combinationwith R¹² together with the atoms to which they are joined, form a ringwhich is a 5-, or 6-membered alicyclic ring, a substituted 5-, or6-membered alicyclic ring, a 5-, or 6-membered heterocyclic ring, or asubstituted 5-, or 6-membered heterocyclic ring;p is 0, 1, 2 or 3;wherein at least one of R¹-R¹² is —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore with the provisothat when at least one of the R¹-R¹² is —(CR₂)_(n)-fluorophore that thefluorophore comprise a nitrogen atom that is covalently bonded to the—(CR₂)_(n)—.

In one aspect exactly one of R¹-R⁸ is —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore, in a further aspect, exactly one of R², R³, R⁶or R⁷ is —(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore. In oneembodiment R and R′ are each hydrogen. The fluorophore can be anyreporter moiety known to one of skill in the art. Such a fluorophoreincludes, but are not limited, to those fluorophores selected from thegroup consisting of dansyl, xanthene, cyanine, borapolyazaindacene,pyrene, naphthalene, coumarin, oxazine and derivatives thereof. Thexanthenes are further classified to include the members selected fromthe group consisting of fluorescein or derivatives thereof, rhodamine orderivatives thereof, rhodol or derivatives thereof, and rosamine orderivatives thereof. In a particular embodiment, the fluorophore is axanthene, particularly fluorescein or a derivative thereof, and thelinker is —(CH₂)_(n)NR′—. In another particular embodiment, thefluorophore is dansyl and the linker is —(CH₂)_(n)NR′—. In analternative embodiment the linker is —(CH₂)_(n)— wherein the fluorophoreis naphthalene. Exemplary compounds include members selected from thegroup consisting of Compound 5, 6, 9, 10, 13, 14 and 15.

The present fluorophores are independently substituted by substituentsselected from the group consisting of hydrogen, halogen, amino,substituted amino, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, sulfo, and reactive group.In one aspect, a xanthene fluorophore is substituted by halogen such asfluorine, chlorine or bromine.

In an exemplary embodiment, the nitrogen substitutents R¹⁵, R¹⁶, R¹⁷ andR¹⁸ are each hydrogen. In a further embodiment, R¹³ and R¹⁴ areindependently hydrogen or a salt ion representing a cell impermeantversion of the present metal ion indicators. In yet another embodimentR¹³ and R¹⁴ are independently —CH₂OCOCH₃ represent a cell permeantversion of the present compounds. Alternatively, R¹³ and R¹⁴ are eachCH₃, also representing a cell permeant version of the present compounds.

The reactive group, solid support and carrier molecule when substitutedon the present compounds comprise a linker that is a single covalentbond, or a covalent linkage that is linear or branched, cyclic orheterocyclic, saturated or unsaturated, having 1-20 nonhydrogen atomsselected from the group consisting of C, N, P, O and S; and are composedof any combination of ether, thioether, amine, ester, carboxamide,sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.

The reactive group is selected from the group consisting of anacrylamide, an activated ester of a carboxylic acid, a carboxylic ester,an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, ananhydride, an aniline, an amine, an aryl halide, an azide, an aziridine,a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine,a hydrazine, an imido ester, an isocyanate, an isothiocyanate, amaleimide, a phosphoramidite, a reactive platinum complex, a silylhalide, a sulfonyl halide, a thiol and a photoactivatable group. In aparticular embodiment the reactive group is selected from the groupconsisting of carboxylic acid, succinimidyl ester of a carboxylic acid,hydrazide, amine and a maleimide.

The carrier molecule is selected from the group consisting of an aminoacid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell or a virus. In a particularembodiment the carrier molecule is selected from the group consisting ofan antibody or fragment thereof, an avidin or streptavidin, a biotin, ablood component protein, a dextran, an enzyme, an enzyme inhibitor, ahormone, an IgG binding protein, a fluorescent protein, a growth factor,a lectin, a lipopolysaccharide, a microorganism, a metal bindingprotein, a metal chelating moiety, a non-biological microparticle, apeptide toxin, a phosphotidylserine-binding protein, a structuralprotein, a small-molecule drug, or a tyramide.

The solid support is selected from the group consisting of amicrofluidic chip, a silicon chip, a microscope slide, a microplatewell, silica gels, polymeric membranes, particles, derivatized plasticfilms, glass beads, cotton, plastic beads, alumina gels,polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon,latex bead, magnetic bead, paramagnetic bead, and superparamagneticbead. In a particular embodiment the solid support is selected from thegroup consisting of Sepharose, poly(acrylate), polystyrene,poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch,FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose,diazocellulose and starch.

In a further embodiment of the present invention, the present compoundsform a composition with a metal ion wherein the composition comprisesany present compound and a metal ion that is capable of being chelatedby the compound.

The present compounds can be utilized to bind, detect, quantitate,monitor and further analyze metal ions. Thus, an exemplary method forbinding a target metal ion in a sample, comprising steps of:

-   -   a. contacting the sample with a present compound to form a        contacted sample; and,    -   b. incubating the contacted sample for a sufficient amount of        time to allow the compound to chelate the target metal ion        whereby the metal ion is bound.

The metal ions that can be bound by the present compounds include, butare not limited to, Ca²⁺, Zn²⁺, Mg²⁺, Ga³⁺, Tb³⁺, La³⁺, Pb²⁺, Hg²⁺,Cd²⁺, Cu²⁺, Ni²⁺, Co²⁺, Fe²⁺, Mn²⁺, Ba²⁺, and Sr²⁺. Particularlyrelevant are those metal ions that are present in biological systemssuch as those selected from the group consisting of Ca²⁺, Mg²⁺, Fe²⁺ andZn²⁺. In an exemplary embodiment, the present compounds are used to bindcalcium ions.

The sample typically is or comprises a biological system wherein thesample is selected from the group consisting of live cells,intracellular fluids, extracellular fluids, biological fluids,biological fermentation media, environmental sample, industrial samples,proteins, peptides, buffer solutions or biological fluids and chemicalreactors. In a further embodiment the sample is selected from the groupconsisting of blood cells, immune cells, cultured cells, muscle tissue,neurons, extracellular vesicles; vascular tissue, blood fluids, saliva,urine; water, soil, waste water, sea water; pharmaceuticals, foodstuffsand beverages.

The present method further comprises detecting a target metal ionwherein the sample is illuminated with an appropriate wavelength wherebythe target metal ion is detected. In this instance the present compoundscomprise a reporter moiety, typically a fluorophore.

In an exemplary embodiment the present compounds are utilized to detectmetal ions in a live cell wherein the compounds comprise a lipophilicgroup such as an AM or acetate ester. In this instance the method fordetecting target ions in a live cell comprises the steps of:

-   -   a) contacting a sample of live cells with a present compound        with the proviso that at least one of R¹³ or R¹⁴ is —CH₂OCOCH₃        or CH₃;    -   b) incubating the sample and the compound for sufficient time to        allow the compound to chelate the target metal ion; and,    -   c) illuminating the sample with an appropriate wavelength to        generate a detectable signal that is a result of PET whereby the        target ion is detected in a live cell.

In this instance the metal ion to be detected includes those membersselected from the group consisting of Hg²⁺, Ni²⁺, ca²⁺, Mg²⁺, Fe²⁺ andZn²⁺.

The present invention also provides kits for binding, detecting,quantitating, monitoring and otherwise analyzing metal ions wherein thekit comprises at least one compound according to the present inventionand instructions for use thereof. In a further embodiment, the kitcomprises one or more components selected from the group consisting of acalibration standard of a metal ion, an ionophore, a metal ion indicatorother than for calcium ions, a detectable signal standard, an aqueousbuffer solution, an antibody or fragment thereof, a reference dyestandard and an organic solvent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows the binding and detection of a titration (0 μM to 39 μM)of calcium ions in solution wherein a Stokes shift of about 220 nm wasobserved. The calcium ion solution containing the present compound wasexcited at a wavelength of 331 nm and the resulting emission wavelengthwas at 549 nm.

FIG. 2: Shows the binding and detection of a titration (0 μM to 39 μM)of calcium ions in solution wherein a Stokes shift of about 120 nm wasobserved. The calcium ion solution containing the present compound wasexcited at a wavelength of 432 nm and the resulting emission wavelengthwas at 550 nm.

FIG. 3: Shows the detection of intracellular calcium ions in live Jurkatcells using a live cell version of the present compound.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The present invention is based upon the phenomenon in which the opticalproperties of a fluorophore can be modulated by strategic covalentattachment of a metal ion-binding moiety (a chelator). In the invention,it has been found that certain ion chelators reduce the fluorescence ofthe fluorophore by a through-space interaction known as PET, in whichfluorescence is inhibited by interaction of the excited statefluorophore with an electron-rich chelator moiety. As the chelatormoiety binds metal ion(s), the PET effect is diminished, resulting inincreased fluorescence from the fluorophore.

Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a metal chelator” includes aplurality of chelators and reference to “a metal ion” includes aplurality of ions and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the invention as described herein.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds of the invention may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose anappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VoGEL's ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and an acyl radical onat least one terminus of the alkane radical. The “acyl radical” is thegroup derived from a carboxylic acid by removing the —OH moietytherefrom.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”.

Exemplary alkyl groups of use in the present invention contain betweenabout one and about twenty five carbon atoms (e.g. methyl, ethyl and thelike). Straight, branched or cyclic hydrocarbon chains having eight orfewer carbon atoms will also be referred to herein as “lower alkyl”. Inaddition, the term “alkyl” as used herein further includes one or moresubstitutions at one or more carbon atoms of the hydrocarbon chainfragment.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P and S, and wherein the nitrogen,phosphorous and sulfur atoms are optionally oxidized, and the nitrogenheteroatom is optionally quaternized. The heteroatom(s) O, N, P, S andSi may be placed at any interior position of the heteroalkyl group or atthe position at which the alkyl group is attached to the remainder ofthe molecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (preferablyfrom 1 to 3 rings), which are fused together or linked covalently. Theterm “heteroaryl” refers to aryl groups (or rings) that contain from oneto four heteroatoms selected from N, O, and S, wherein the nitrogen andsulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2 m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR′″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P) and silicon (Si).

The term “amino” or “amine group” refers to the group —NR′R″ (orN⁺RR′R″) where R, R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substitutedheteroaryl. A substituted amine being an amine group wherein R′ or R″ isother than hydrogen. In a primary amino group, both R′ and R″ arehydrogen, whereas in a secondary amino group, either, but not both, R′or R″ is hydrogen. In addition, the terms “amine” and “amino” caninclude protonated and quaternized versions of nitrogen, comprising thegroup —N⁺RR′R″ and its biologically compatible anionic counterions.

The term “affinity” as used herein refers to the strength of the bindinginteraction of two molecules, such as a metal chelating compound and ametal ion or a positively charged moiety and a negatively chargedmoiety.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “carrier molecule” as used herein refers to a compound of thepresent invention that is covalently bonded to a biological or anon-biological component. Such components include, but are not limitedto, an amino acid, a peptide, a protein, a polysaccharide, a nucleoside,a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen,a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof.

The term “cell permeable” as used herein refers to compounds of thepresent invention that are able to cross the cell membrane of livecells. Lipophilc groups that are covalently attached to the presentcompounds, facilitate this permeability and live cell entry. Once insidethe cells, the lipophilic groups are hydrolyzed resulting in chargedmolecules that are well retained in living cells. Particularly usefullipophilic groups include acetoxymethyl (AM) ester and acetate esterswherein once inside the cells the groups are cleaved by nonspecificesterases resulting in charged molecules.

The term “complex” as used herein refers to the association of two ormore molecules, usually by non-covalent bonding.

The term “detectable response” as used herein refers to a change in oran occurrence of, a signal that is directly or indirectly detectableeither by observation or by instrumentation and the presence ormagnitude of which is a function of the presence of a target metal ionin the test sample. Typically, the detectable response is an opticalresponse resulting in a change in the wavelength distribution patternsor intensity of absorbance or fluorescence or a change in light scatter,fluorescence quantum yield, fluorescence lifetime, fluorescencepolarization, a shift in excitation or emission wavelength or acombination of the above parameters. The detectable change in a givenspectral property is generally an increase or a decrease. However,spectral changes that result in an enhancement of fluorescence intensityand/or a shift in the wavelength of fluorescence emission or excitationare also useful. The change in fluorescence on ion binding is usuallydue to conformational or electronic changes in the indicator that mayoccur in either the excited or ground state of the fluorophore, due tochanges in electron density at the ion binding site, due to quenching offluorescence by the bound target metal ion, or due to any combination ofthese or other effects. Alternatively, the detectable response is anoccurrence of a signal wherein the fluorophore is inherently fluorescentand does not produce a change in signal upon binding to a metal ion orbiological compound.

The term “fluorophore” as used herein refers to a composition that isinherently fluorescent or demonstrates a change in fluorescence uponbinding to a biological compound or metal ion, or metabolism by anenzyme, i.e., fluorogenic. Fluorophores may be substituted to alter thesolubility, spectral properties or physical properties of thefluorophore. Numerous fluorophores are known to those skilled in the artand include, but are not limited to coumarin, acridine, furan, dansyl,cyanine, pyrene, naphthalene, benzofurans, quinolines, quinazolinones,indoles, benzazoles, borapolyazaindacenes, oxazine and xanthenes, withthe latter including fluoresceins, rhodamines, rosamine and rhodols aswell as other fluorophores described in RICHARD P. HAUGLAND, MOLECULARPROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (9^(th)edition, including the CD-ROM, Sep. 2002). The fluorophore moiety may besubstituted by substituents that enhance solubility, live cellpermeability and alter spectra absorption and emission.

The term “kit” as used refers to a packaged set of related components,typically one or more compounds or compositions.

The term “metal chelator” or “metal chelating compound” as used hereinrefers to a chemical compound that combines with a metal ion to form achelate ring structure.

The term “metal ion” or “target metal ion” as used herein refers to anymetal cation that is capable of being chelated by the present BAPTAmetal chelating compound. Typically, these metal ions are physiologicaland or nutritional relevant metal ion such as Na⁺, K⁺, Zn²⁺, Mg²⁺, Fe²⁺,and Ca²⁺. The term metal ion used herein also refers to the metal ionsGa³⁺, Tb³⁺, La³⁺, Pb²⁺, Hg²⁺, cd²⁺, Cu²⁺, Ni²⁺, Co²⁺, Mn²⁺, Ba²⁺, andSr²⁺.

The term “photoinduced electron transfer (PET)” as used herein refers tointramolecular electron transfer.

The terms “protein” and “polypeptide” are used herein in a generic senseto include polymers of amino acid residues of any length. The term“peptide” as used herein refers to a polymer in which the monomers areamino acids and are joined together through amide bonds, alternativelyreferred to as a polypeptide. When the amino acids are α-amino acids,either the L-optical isomer or the D-optical isomer can be used.Additionally, unnatural amino acids, for example, β-alanine,phenylglycine and homoarginine are also included. Commonly encounteredamino acids that are not gene-encoded may also be used in the presentinvention. All of the amino acids used in the present invention may beeither the D- or L-isomer. The L-isomers are generally preferred. Inaddition, other peptidomimetics are also useful in the presentinvention. For a general review, see, Spatola, A. F., in Chemistry andBiochemistry of Amino Acids, Peptides and Proteins, B. Weinstein, eds.,Marcel Dekker, New York, p. 267 (1983).

The term “reactive group” as used herein refers to a group that iscapable of reacting with another chemical group to form a covalent bond,i.e. is covalently reactive under suitable reaction conditions, andgenerally represents a point of attachment for another substance. Thereactive group is a moiety, such as carboxylic acid or succinimidylester, on the compounds of the present invention that is capable ofchemically reacting with a functional group on a different compound toform a covalent linkage. Reactive groups generally include nucleophiles,electrophiles and photoactivatable groups.

Exemplary reactive groups include, but are not limited to, olefins,acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes,ketones, carboxylic acids, esters, amides, cyanates, isocyanates,thiocyanates, isothiocyanates, amines, hydrazines, hydrazones,hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides,disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids,acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles,amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamicacids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines,ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates,imines, azides, azo compounds, azoxy compounds, and nitroso compounds.Reactive functional groups also include those used to preparebioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and thelike. Methods to prepare each of these functional groups are well knownin the art and their application to or modification for a particularpurpose is within the ability of one of skill in the art (see, forexample, Sandler and Karo, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS,Academic Press, San Diego, 1989).

The term “sample” as used herein refers to any material that may containtarget metal ions, as defined above. Typically, the sample is a livecell or a biological fluid that comprises endogenous host cell proteins.Alternatively, the sample may be a buffer solution or an environmentalsample containing target metal ions. The sample may be in an aqueoussolution, a viable cell culture or immobilized on a solid or semi solidsurface such as a polyacrylamide gel, membrane blot or on a microarray.

The term “Stokes shift” as used herein refers to the difference inwavelength between absorbed and emitted energy. Specifically, the Stokesshift is the difference (usually in frequency units) between thespectral positions and the band maxima (or band origin) of theabsorption and luminescence arising from the same electronictransitions.

The Compounds

In general, for ease of understanding the present invention, the metalion binding compounds and corresponding substituents will first bedescribed in detail, followed by the many and varied methods in whichthe compounds find uses, which is followed by exemplified methods of useand synthesis of certain novel compounds that are particularlyadvantageous for use with the methods of the present invention.

The present compounds find utility in binding target metal ions in asample. The sample includes live cells or a biological fluid thatcomprises endogenous host cell proteins, buffer solutions andenvironmental samples. Therefore, the present compounds, when comprisinga fluorophore or fluorescent protein moiety (reporter moiety), findutility in binding, isolating, quantitating, monitoring and detectingtarget metal ions wherein the detectable signal is modulated byphotoinduced electron transfer (PET). Detection of target metal ions canalso be accomplished in live cells wherein the present compoundcomprises a lipophilic group such as an AM or acetate ester that allowsfor entry across the live cell membrane. Once inside the cellsnonspecific esterases cleave the AM or acetate ester resulting in acharged molecule that is well retained in the cell. These presentcompounds are particularly useful for binding physiologically relevantlevels of calcium.

The present compounds consist of three functional elements, the ionsensing moiety (chelating moiety), the reporter moiety (fluorophore orfluorescent protein) and spacer or linker between the sensing andreporter moieties of the present compound that provides for photoinducedelectron transfer (PET) upon binding of a metal ion and excitation by anappropriate wavelength. The distinguishing feature of these compounds isthis linker, —(CR₂)_(n)NR′—, wherein the alkyl spacer prevents directthrough-bond conjugation between the electron orbitals of the metalchelator and the reporter moiety. In this instance, as used herein“conjugation” refers to the sharing of pi-electrons between alternatingpi orbitals. Thus, the metal chelator and reporter moiety are notconjugated, whereas other known chelators are conjugated to the reportermoiety resulting in a detectable signal that is not modulated by PET.This disruption of direct conjugation results in non-fluorescentcompounds until a metal ion is bound by an appropriate metal ion and thecompound is illuminated by an appropriate wavelength. Alternatively, thelinker is —(CR₂)_(n)— wherein the reporter moiety comprises a nitrogenatom that is covalently bonded to a carbon atom of the linker.

Therefore, the present compounds for the detection of metal ions whereina detectable response is a result of photoinduced electron transfer(PET), and the compound comprises a metal chelating moiety and afluorophore or a fluorescent protein that is covalently bonded to themetal chelating moiety by linker —(CR₂)_(n)NR′— or —(CR₂)_(n)— wherein Rand R′ are independently selected from the group consisting of hydrogen,alkyl, and substituted alkyl and n is 1-10. However, when the linker is—(CR₂)_(n)— the terminal carbon atom must be directly and covalentlybonded to a nitrogen atom of the fluorophore.

In a more specific embodiment, the present compound binds and detectscalcium ions wherein a detectable response is a result of photoinducedelectron transfer (PET) and the compound comprises a metal chelatingmoiety and a fluorophore or a fluorescent protein that is covalentlybonded to the metal chelating moiety by linker —(CR₂)_(n)NR′— or—(CR₂)_(n)— wherein R and R′ are independently selected from the groupconsisting of hydrogen, alkyl, and substituted alkyl and n is 1-10.Again, when the linker is —(CR₂)_(n)— the terminal carbon atom of thelinker must be directly and covalently bonded to a nitrogen atom of thefluorophore.

In all cases the present compounds exhibit a Stokes shift that isgreater than about 50 nm, more preferably greater than about 100 nm,even more preferably greater than about 150 nm. In some instances thepresent compounds exhibit a Stokes shift greater than about 200 nm andin particularly desirable embodiments, the present compounds exhibit aStokes shift greater than about 250 nm. This larger Stokes shift isadvantageous for distinguishing the detectable signal fromautofluorescence of biological samples and for the application ofmulticolor assays allowing for the simultaneous detection of multipleanalytes. To the best of our knowledge the present compounds representthe only metal ion sensing compounds with a Stokes shift greater thanabout 50 nm, which represents a major improvement over known andcommonly used metal ion indicators. This is particular advantageous forthe detection of intracellular calcium ion concentrations.

Chelating Moiety

The ion-sensing or chelating moiety of the present compound is anymoiety that will bind or chelate metal ions. Typically this results in achange in the fluorescent signal. Metal ions of the present invention,include but are not limited to, Ca²⁺, Zn²⁺, Mg²⁺, Ga³⁺, Tb³⁺, La³⁺,Pb²⁺, Hg²⁺, Cd²⁺, Cu²⁺, Ni²⁺, Co²⁺, Fe²⁺, Mn²⁺, Ba²⁺, and Sr²⁺. In oneaspect the metal ion is a physiological relevant ion selected from thegroup consisting of Ca²⁺, Mg²⁺, Fe²⁺ and Zn²⁺. In a further aspect themetal ion is Ca²⁺, which is most notably chelated by the well-knownBAPTA chelating moiety.

The term “BAPTA” as used herein refers to a metal-chelating compoundthat is 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or itsanalogs, derivatives, ring-fused variants and conjugates, and allmetallic and nonmetallic salts, partial salts and hydrates thereof,including any corresponding compounds disclosed in U.S. Pat. Nos.4,603,209; 4,849,362; 5,049,673; 5,453,517; 5,459,276; 5,516,911;5,501,980; 6,162,931 and 5,773,227. When used generically, “BAPTA”refers to two benzene rings that are joined by a C₁-C₃ hydrocarbonbridge terminated by oxygen atoms, including methylenedioxy (—OCH₂O—),ethylenedioxy (—OCH₂CH₂O—) or propylenedioxy (—OCH₂CH₂CH₂O—) bridginggroups, where each benzene ring is optionally substituted by one or moresubstituents that adjust the metal ion-binding affinity, solubility,chemical reactivity, spectral properties or other physical properties ofthe compound. BAPTA derivatives additionally include compounds in whichthe benzene rings of the BAPTA structure are substituted by or fused toadditional aromatic, or heteroaromatic rings.

Thus, in one aspect the chelating moiety of the present compounds isrepresented by the formula:

wherein R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are independently hydrogen (H) or C₁-C₆alkyl and R¹³ and R¹⁴ are independently hydrogen (H), C₁-C₆ alkyl,—CH₂OCO(alkyl) or a salt ion. In one aspect R¹⁵, R¹⁶, R¹⁷ and R¹⁸ areeach hydrogen. When R¹³ and R¹⁴ are independently —CH₂OCOCH₃ or CH₃ thepresent compounds are utilized as live cell metal ion indicators.Alternatively, R¹³ and R¹⁴ are independently hydrogen or a salt ionwherein the resulting compounds are not capable of passively crossingthe live cell membrane.

Thus, cell impermeant versions of the present compounds are, in oneaspect, have the following formula:

The cell impermeant version of the compounds may be synthesized in thismanner to be used for detection of metal ions in an environment otherthan live cells or the cell impermeant version may be a result of thecell permeant version that has been loaded in to live cells andsubsequently cleaved by an intracellular enzyme. These cell permeantversions of the compounds are thus converted to cell impermeant versionin the cells resulting in metal ion indicators that are well retained inlive cells.

In one aspect, the live-cell versions of the present compounds have thefollowing formula:

Wherein the present compound comprises a substrate for a non-specificester, typically the compound comprises an AM ester wherein R¹³ and/orR¹⁴ are —CH₂OCOCH₃.

In another aspect, the compounds are according to the following formula:

wherein the compound comprises an acetate group.

The benzene substituents, R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ aretypically substituted by substitutents well known in the art for BAPTAcompounds. These substituents are selected independently from the groupconsisting of hydrogen, halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃),hydroxyl (—OH), C₂-C₆ alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl,heteroaryl, amino (−) NR¹⁹R²⁰, aldehyde, carboxyl, azido, nitro,nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule,solid support, reporter molecule, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore and —(CR₂)_(n)-fluorophore. Alternatively, anytwo adjacent ring substituents in combination constitute a cyclicsubstituent that is substituted or unsubstituted cycloalkyl,cycloheteroalkyl, aryl, fused aryl, heteroaryl or fused heteroaryl. Inone aspect a member selected from R¹ in combination with R²; R² incombination with R³; R³ in combination with R⁴; R⁵ in combination withR⁶; R⁶ in combination with R⁷; and R⁷ in combination with R⁸ togetherwith the atoms to which they are joined, form a ring which is a 5-, 6-or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl,a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or asubstituted 5-, 6- or 7-membered heteroaryl.

The amino substituents, R¹⁹ and R²⁰, are independently selected from thegroup consisting of H, C₁-C₆ alkyl, substituted alkyl, C₁-C₆carboxyalkyl (—(CH₂)₁₋₆COOR¹³), an alpha-acyloxyalkyl, a biologicallycompatible salt, aryl, substituted aryl, aryl alkyl, substituted arylalkyl, heteroaryl, and substituted heteroaryl.

The linker substituents, R, R′ and R″, are independently selected fromthe group consisting of hydrogen, alkyl, and substituted alkyl wherein nis 1-10. In addition, R″ is also independently selected from the groupconsisting of a fluorophore, carrier molecule, solid support andreactive group.

The bridge substituents, R⁹, R¹⁰, R¹¹ and R¹², are independentlyselected from the group consisting of hydrogen, a reactive group, acarrier molecule, a solid support, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore, —(CR₂)_(n)-fluorophore and C₁-C₆ alkylwherein p is 0, 1, 2 or 3. Alternatively, adjacent substituents R⁹ andR¹⁰ or R¹¹ and R¹², taken in combination, constitute a 5-membered or6-membered alicyclic or heterocyclic ring. In one aspect a memberselected from R⁹ in combination with R¹⁰; or R¹¹ in combination with R¹²together with the atoms to which they are joined, form a ring which is a5-, or 6-membered alicyclic ring, a substituted 5-, or 6-memberedalicyclic ring, a 5-, or 6-membered heterocyclic ring, or a substituted5-, or 6-membered heterocyclic ring.

The present compounds comprise at least one linker or linker covalentlybonded to a fluorophore wherein the detectable response is a result ofphotoinduced electron transfer (PET). In one aspect the linker has theformula —(CR₂)_(n)NR′R″. When covalently bonded to a fluorophore thelinker and reporter moiety have the formula —(CR₂)_(n)NR′-fluorophore.Alternatively the linker is an C₁-C₁₀ alkyl group wherein a terminalcarbon atom is covalently bonded to a nitrogen atom of a reporter moietyhaving the formula —(CR₂)_(n)-fluorophore, wherein n is 1-10 for alllinker formulas.

Thus, when the present compounds comprise a chelating moiety accordingto formula I at least one of R₁-R₁₂ is —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore. Typically, thelinker is attached to a benzene substituent wherein at least one ofR¹-R⁸ is —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore. More typically at least one of R³ or R⁶ is apresent linker.

Therefore, in one aspect, the present compounds have the formula:

wherein R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵,R¹⁶, R¹⁷ and R¹⁸ are as previously defined. In a further aspect, R⁵, R⁶,R⁷ and R⁸ are independently hydrogen, sulfo, alkyl or halogen, whereinthe halogen is typically fluorine, chlorine or bromine. In yet a furtheraspect, when R³ is or is attached to a present linker R⁶ is typically analkyl or halogen. Preferably the alkyl is methyl.

Reporter Moiety

The reporter moiety of the present invention functions as a reportermolecule to confer a detectable signal, directly or indirectly, to thetarget metal ions. This results in the ability to detect, monitor andquantitate target metal ions in a sample.

The present reporter molecules can be any reporter molecule known to oneskilled in the art. A wide variety of chemically reactive fluorescentdyes that may be suitable for incorporation into the compounds of theinvention are already known in the art (RICHARD P. HAUGLAND, MOLECULARPROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS (2002)).Reporter moieties include, without limitation, a fluorophore, afluorescent protein, or a tandem dye (energy transfer pair). Preferably,the reporter moiety is a fluorophore wherein when the present compoundsare non-fluorescent until bound by a metal ion. After binding a metalion and upon illumination with an appropriate wavelength the compoundproduces a detectable signal modulated by PET.

In one embodiment, at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R¹⁰, R¹¹ and R¹² is attached to a reporter moiety via a present linker.In a particular aspect at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, andR⁸ is attached to a reporter moiety. In a preferred aspect, either R³ orR⁶ is attached to a reporter moiety.

Where the detectable response is a fluorescence response, it istypically a change in fluorescence, such as a change in the intensity,excitation or emission wavelength, distribution of fluorescence,fluorescence lifetime, fluorescence polarization, or a combinationthereof. Preferably, the detectable optical response upon binding atarget ion is a change in fluorescence intensity that is greater thanapproximately 150% relative to the same compound in the absence of themetal ion, more preferably greater than 5-fold, and most preferably morethat 10-fold. In combination with the large Stokes shift of thecompounds of the present invention, this large increase in fluorescentsignal over baseline has not been previously observed with other calciumindicators that comprise a fluorescent PET signal mechanism. In anotheraspect, the detectable optical response upon binding the target metalion is a shift in either the maximal excitation or emission wavelengthor both that is greater than about 50 nm, more preferably greater thanabout 100 nm.

A fluorescent dye of the present invention is any chemical moiety thatexhibits an absorption maximum beyond 280 nm, and when covalently linkedto a metal chelating moiety of the present invention, forms a presentfluorogenic metal ion-binding compound. A preferred embodiment fordetecting calcium ions in live cells or calcium ions secreted from livecells is a fluorogenic calcium-binding compound wherein the reportermoiety is fluorophore. As described above, the linker is —(CH₂)_(n)NR′—or —(CH₂)_(n)—.

Dyes of the present invention include, without limitation; a pyrene, ananthracene, a naphthalene, an acridine, a stilbene, an indole orbenzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a carbocyanine (includingany corresponding compounds in U.S. Ser. Nos. 09/557,275; 09/968,401 and09/969,853 and U.S. Pat. Nos. 6,403,807; 6,348,599; 5,486,616;5,268,486; 5,569,587; 5,569,766; 5,627,027 and 6,048,982), acarbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, aperylene, a pyridine, a quinoline, a borapolyazaindacene (including anycorresponding compounds disclosed in U.S. Pat. Nos. 4,774,339;5,187,288; 5,248,782; 5,274,113; and 5,433,896), a xanthene (includingany corresponding compounds disclosed in U.S. Pat. Nos. 6,162,931;6,130,101; 6,229,055; 6,339,392; 5,451,343 and U.S. Ser. No.09/922,333), an oxazine or a benzoxazine, a carbazine (including anycorresponding compounds disclosed in U.S. Pat. No. 4,810,636), aphenalenone, a coumarin (including an corresponding compounds disclosedin U.S. Pat. Nos. 5,696,157; 5,459,276; 5,501,980 and 5,830,912), abenzofuran (including an corresponding compounds disclosed in U.S. Pat.Nos. 4,603,209 and 4,849,362) and benzphenalenone (including anycorresponding compounds disclosed in U.S. Pat. No. 4,812,409) andderivatives thereof. As used herein, oxazines include resorufins(including any corresponding compounds disclosed in U.S. Pat. No.5,242,805), aminooxazinones, diaminooxazines, and theirbenzo-substituted analogs.

Where the dye is a xanthene, the dye is optionally a fluorescein, arhodol (including any corresponding compounds disclosed in U.S. Pat.Nos. 5,227,487 and 5,442,045), a rosamine or a rhodamine (including anycorresponding compounds in U.S. Pat. Nos. 5,798,276; 5,846,737;5,847,162; 6,017,712; 6,025,505; 6,080,852; 6,716,979; 6,562,632). Asused herein, fluorescein includes benzo- or dibenzofluoresceins,seminaphthofluoresceins, or naphthofluoresceins. Similarly, as usedherein rhodol includes seminaphthorhodafluors (including anycorresponding compounds disclosed in U.S. Pat. No. 4,945,171).Fluorinated xanthene dyes have been described previously as possessingparticularly useful fluorescence properties (Int. Publ. No. WO 97/39064and U.S. Pat. No. 6,162,931).

Preferred dyes of the invention include dansyl, xanthene, cyanine,borapolyazaindacene, pyrene, naphthalene, coumarin, oxazine andderivatives thereof. Preferred xanthenes are fluorescein, rhodamine andderivatives thereof, naphthalene and dansyl.

Typically the dye contains one or more aromatic or heteroaromatic rings,that are optionally substituted one or more times by a variety ofsubstituents, including without limitation, halogen, nitro, sulfo,cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,arylalkyl, acyl, aryl or heteroaryl ring system, benzo, or othersubstituents typically present on chromophores or fluorophores known inthe art.

In an exemplary embodiment, the dyes are independently substituted bysubstituents selected from the group consisting of hydrogen, halogen,amino, substituted amino, alkyl, substituted alkyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, alkoxy, sulfo, reactive groupand carrier molecule. In another embodiment, the xanthene dyes of thisinvention comprise both compounds substituted and unsubstituted on thecarbon atom of the central ring of the xanthene by substituentstypically found in the xanthene-based dyes such as phenyl andsubstituted-phenyl moieties. Most preferred dyes are rhodamine,fluorescein, dansyl, naphthalene and derivatives thereof. The choice ofthe dye attached to the chelating moiety will determine the metalion-binding compound's absorption and fluorescence emission propertiesas well as its live cell properties, i.e. ability to localize tomitochondria.

Selected sulfonated reporter moieties also exhibit advantageousproperties, and include sulfonated pyrenes, coumarins, carbocyanines,and xanthenes (as described in U.S. Pat. Nos. 5,132,432; 5,696,157;5,268,486; 6,130,101). Sulfonated pyrenes and coumarins are typicallyexcited at wavelengths below about 450 nm (U.S. Pat. Nos. 5,132,432 and5,696,157).

In an exemplary embodiment, the dye has a Stokes shift larger than about50 nm. In a particularly useful embodiment, the dye has a Stokes shiftlarger than about 100 nm, more preferably larger than about 150 nm. In afurther embodiment, the present compounds have a Stokes shift largerthan about 200 nm, more preferably larger than about 250 nm.

Fluorescent proteins also find use as reporter moieties for the chelatecompounds of the present invention. Examples of fluorescent proteinsinclude green fluorescent protein (GFP) and the phycobiliproteins andthe derivatives thereof. The fluorescent proteins, especiallyphycobiliproteins, are particularly useful for creating tandemdye-reporter molecules. These tandem dyes comprise a fluorescent proteinand a fluorophore for the purposes of obtaining a larger Stokes shift,wherein the emission spectra are farther shifted from the wavelength ofthe fluorescent protein's absorption spectra. This property isparticularly advantageous for detecting a low quantity of a target ionin a sample wherein the emitted fluorescent light is maximallyoptimized; in other words, little to none of the emitted light isreabsorbed by the fluorescent protein. For this to work, the fluorescentprotein and fluorophore function as an energy transfer pair wherein thefluorescent protein emits at the wavelength that the acceptorfluorophore absorbs and the fluorophore then emits at a wavelengthfarther from the fluorescent proteins than could have been obtained withonly the fluorescent protein. Alternatively, the fluorophore functionsas the energy donor and the fluorescent protein is the energy acceptor.Particularly useful fluorescent proteins are the phycobiliproteinsdisclosed in U.S. Pat. Nos. 4,520,110; 4,859,582; 5,055,556 and thefluorophore bilin protein combinations disclosed in U.S. Pat. No.4,542,104. Alternatively, two or more fluorophore dyes can function asan energy transfer pair wherein one fluorophore is a donor dye and theother is the acceptor dye including any dye compounds disclosed in U.S.Pat. Nos. 6,358,684; 5,863,727; 6,372,445; 6,221,606; 6,008,379;5,945,526; 5,863,727; 5,800,996; 6,335,440; 6,008,373; 6,184,379;6,140,494 and 5,656,554.

The reactive group, carrier molecules, and solid support comprise alinker that is used to covalently attach the substituents to thechelating moiety or reporter moiety of the present compounds. The solidsupport, carrier molecule or reactive group may be directly attached(where Linker is a single bond) to the moieties or attached through aseries of stable bonds. When the linker is a series of stable covalentbonds the linker typically incorporates 1-30 nonhydrogen atoms selectedfrom the group consisting of C, N, O, S and P. When the linker is not asingle covalent bond, the linker may be any combination of stablechemical bonds, optionally including, single, double, triple or aromaticcarbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen-nitrogenbonds, carbon-oxygen bonds, sulfur-sulfur bonds, carbon-sulfur bonds,phosphorus-oxygen bonds, phosphorus-nitrogen bonds, andnitrogen-platinum bonds. Typically the linker incorporates less than 15nonhydrogen atoms and are composed of any combination of ether,thioether, thiourea, amine, ester, carboxamide, sulfonamide, hydrazidebonds and aromatic or heteroaromatic bonds. Typically the linker is acombination of single carbon-carbon bonds and carboxamide, sulfonamideor thioether bonds. The bonds of the linker typically result in thefollowing moieties that can be found in the linker: ether, thioether,carboxamide, thiourea, sulfonamide, urea, urethane, hydrazine, alkyl,aryl, heteroaryl, alkoky, cycloalkyl and amine moieties. Examples of alinker include substituted or unsubstituted polymethylene, arylene,alkylarylene, arylenealkyl, or arylthio.

In one embodiment, the linker contains 1-6 carbon atoms; in another, thelinker comprises a thioether linkage. Exemplary linking members includea moiety that includes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, and the like.In another embodiment, the linker is or incorporates the formula—(CH₂)_(d)(CONH(CH₂)_(e))_(z)— or where d is an integer from 0-5, e isan integer from 1-5 and z is 0 or 1. In a further embodiment, the linkeris or incorporates the formula —O—(CH₂)—. In yet another embodiment, thelinker is or incorporates a phenylene or a 2-carboxy-substitutedphenylene.

Any combination of linkers may be used to attach the carrier molecule,solid support or reactive group and the present compounds together. Thelinker may also be substituted to alter the physical properties of thereporter moiety or chelating moiety, such as spectral properties of thedye.

Another important feature of the linker is to provide an adequate spacebetween the carrier molecule, reactive group or solid support and thechelating moiety or reporter moiety so as to prevent steric hinderance.Therefore, the linker of the present compound is important for (1)attaching the carrier molecule, reactive group or solid support to thecompound, (2) providing an adequate space between the carrier molecule,reactive group or solid support and the compound so as not to stericallyhinder the action of the compound and (3) for altering the physicalproperties of the present compounds.

Reactive Groups

In another exemplary embodiment of the invention, the present compoundsare chemically reactive, and are substituted by at least one reactivegroup. The reactive group functions as the site of attachment foranother moiety, such as a carrier molecule or a solid support, whereinthe reactive group chemically reacts with an appropriate reactive orfunctional group on the carrier molecule or solid support. Thus, inanother aspect of the present invention the compounds comprise thechelating moiety, linker, reporter moiety, a reactive group moiety andoptionally a carrier molecule and/or a solid support.

In an exemplary embodiment, the compounds of the invention furthercomprise a reactive group which is a member selected from an acrylamide,an activated ester of a carboxylic acid, a carboxylic ester, an acylazide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, ananiline, an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a photoactivatable group, a reactive platinum complex,a silyl halide, a sulfonyl halide, and a thiol. In a particularembodiment the reactive group is selected from the group consisting ofcarboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide,amine and a maleimide. In exemplary embodiment, at least one memberselected from R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹² is areactive group. Preferably, at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷,and R⁸ is a reactive group, most preferred is at least one of R⁵, R⁶, R⁷or R⁸. Alternatively, if the present compound comprises a carriermolecule or solid support a reactive group may be covalently attachedindependently to those substituents, allowing for further conjugation toa reporter molecule, carrier molecule or solid support.

In one aspect, the compound comprises at least one reactive group thatselectively reacts with an amine group. This amine-reactive group isselected from the group consisting of succinimidyl ester, sulfonylhalide, tetrafluorophenyl ester and iosothiocyanates. Thus, in oneaspect, the present compounds form a covalent bond with an aminecontaining molecule in a sample. In another aspect, the compoundcomprises at least one reactive group that selectively reacts with athiol group. This thiol-reactive group is selected from the groupconsisting of maleimide, haloalkyl and haloacetamide (including anyreactive groups disclosed in U.S. Pat. No. 5,362,628; U.S. Pat. No.5,352,803 and U.S. Pat. No. 5,573,904).

These reactive groups are synthesized during the formation of thepresent compound and carrier molecule and solid support containingcompounds to provide chemically reactive metal ion-binding compounds. Inthis way, compounds incorporating a reactive group can be covalentlyattached to a wide variety of carrier molecules or solid supports thatcontain or are modified to contain functional groups with suitablereactivity, resulting in chemical attachment of the components. In anexemplary embodiment, the reactive group of the compounds of theinvention and the functional group of the carrier molecule or solidsupport comprise electrophiles and nucleophiles that can generate acovalent linkage between them. Alternatively, the reactive groupcomprises a photoactivatable group, which becomes chemically reactiveonly after illumination with light of an appropriate wavelength.Typically, the conjugation reaction between the reactive group and thecarrier molecule or solid support results in one or more atoms of thereactive group being incorporated into a new linkage attaching thepresent compound of the invention to the carrier molecule or solidsupport. Selected examples of functional groups and linkages are shownin Table 1, where the reaction of an electrophilic group and anucleophilic group yields a covalent linkage.

TABLE 1 Examples of some routes to useful covalent linkagesElectrophilic Group Nucleophilic Group Resulting Covalent Linkageactivated esters* amines/anilines carboxamides acrylamides thiolsthioethers acyl azides** amines/anilines carboxamides acyl halidesamines/anilines carboxamides acyl halides alcohols/phenols esters acylnitriles alcohols/phenols esters acyl nitriles amines/anilinescarboxamides aldehydes amines/anilines imines aldehydes or ketoneshydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkylhalides amines/anilines alkyl amines alkyl halides carboxylic acidsesters alkyl halides thiols thioethers alkyl halides alcohols/phenolsethers alkyl sulfonates thiols thioethers alkyl sulfonates carboxylicacids esters alkyl sulfonates alcohols/phenols ethers anhydridesalcohols/phenols esters anhydrides amines/anilines carboxamides arylhalides thiols thiophenols aryl halides amines aryl amines aziridinesthiols thioethers boronates glycols boronate esters carbodiimidescarboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic acidsesters epoxides thiols thioethers haloacetamides thiols thioethershaloplatinate amino platinum complex haloplatinate heterocycle platinumcomplex haloplatinate thiol platinum complex halotriazinesamines/anilines aminotriazines halotriazines alcohols/phenols triazinylethers halotriazines thiols triazinyl thioethers imido estersamines/anilines amidines isocyanates amines/anilines ureas isocyanatesalcohols/phenols urethanes isothiocyanates amines/anilines thioureasmaleimides thiols thioethers phosphoramidites alcohols phosphite esterssilyl halides alcohols silyl ethers sulfonate esters amines/anilinesalkyl amines sulfonate esters thiols thioethers sulfonate esterscarboxylic acids esters sulfonate esters alcohols ethers sulfonylhalides amines/anilines sulfonamides sulfonyl halides phenols/alcoholssulfonate esters *Activated esters, as understood in the art, generallyhave the formula —COΩ, where Ω is a good leaving group (e.g.,succinimidyloxy (—OC₄H₄O₂) sulfosuccinimidyloxy(—OC₄H₃O₂—SO₃H),-1-oxybenzotriazolyl (—OC₆H₄N₃); or an aryloxy group oraryloxy substituted one or more times by electron withdrawingsubstituents such as nitro, fluoro, chloro, cyano, or trifluoromethyl,or combinations thereof, used to form activated aryl esters; or acarboxylic acid activated by a carbodiimide to form an anhydride ormixed anhydride —OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a) and R^(b),which may be the same or different, are C₁-C₆ alkyl, C₁-C₆perfluoroalkyl, or C₁-C₆ alkoxy; or cyclohexyl, 3-dimethylaminopropyl,or N-morpholinoethyl). **Acyl azides can also rearrange to isocyanates

Choice of the reactive group used to attach the compound of theinvention to the substance to be conjugated typically depends on thereactive or functional group on the substance to be conjugated and thetype or length of covalent linkage desired. The types of functionalgroups typically present on the organic or inorganic substances(biomolecule or non-biomolecule) include, but are not limited to,amines, amides, thiols, alcohols, phenols, aldehydes, ketones,phosphates, imidazoles, hydrazines, hydroxylamines, disubstitutedamines, halides, epoxides, silyl halides, carboxylate esters, sulfonateesters, purines, pyrimidines, carboxylic acids, olefinic bonds, or acombination of these groups. A single type of reactive site may beavailable on the substance (typical for polysaccharides or silica), or avariety of sites may occur (e.g., amines, thiols, alcohols, phenols), asis typical for proteins.

Typically, the reactive group will react with an amine, a thiol, analcohol, an aldehyde, a ketone, or with silica. Preferably, reactivegroups react with an amine or a thiol functional group, or with silica.In one embodiment, the reactive group is an acrylamide, an activatedester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde,an alkyl halide, a silyl halide, an anhydride, an aniline, an arylhalide, an azide, an aziridine, a boronate, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine (including hydrazides), animido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a sulfonyl halide, or athiol group. By “reactive platinum complex” is particularly meantchemically reactive platinum complexes such as described in U.S. Pat.No. 5,714,327.

Where the reactive group is an activated ester of a carboxylic acid,such as a succinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester or an isothiocyanates, the resulting compound isparticularly useful for preparing conjugates of carrier molecules suchas proteins, nucleotides, oligonucleotides, or haptens. Where thereactive group is a maleimide, haloalkyl or haloacetamide (including anyreactive groups disclosed in U.S. Pat. No. 5,362,628; U.S. Pat. No.5,352,803 and U.S. Pat. No. 5,573,904 (supra)) the resulting compound isparticularly useful for conjugation to thiol-containing substances.Where the reactive group is a hydrazide, the resulting compound isparticularly useful for conjugation to periodate-oxidized carbohydratesand glycoproteins, and in addition is an aldehyde-fixable polar tracerfor cell microinjection. Where the reactive group is a silyl halide, theresulting compound is particularly useful for conjugation to silicasurfaces, particularly where the silica surface is incorporated into afiber optic probe subsequently used for remote ion detection orquantitation.

In a particular aspect, the reactive group is a photoactivatable groupsuch that the group is only converted to a reactive species afterillumination with an appropriate wavelength. An appropriate wavelengthis generally a UV wavelength that is less than 400 nm. This methodprovides for specific attachment to only the target molecules, either insolution or immobilized on a solid or semi-solid matrix.Photoactivatable reactive groups include, without limitation,benzophenones, aryl azides and diazirines.

Preferably, the reactive group is a photoactivatable group, succinimidylester of a carboxylic acid, a haloacetamide, haloalkyl, a hydrazine, anisothiocyanate, a maleimide group, an aliphatic amine, a silyl halide, acadaverine or a psoralen. More preferably, the reactive group is asuccinimidyl ester of a carboxylic acid, a maleimide, an iodoacetamide,or a silyl halide. In a particular embodiment the reactive group is asuccinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester, an iosothiocyanates or a maleimide.

Carrier Molecules

In an exemplary embodiment, the present compound is covalently bound toa carrier molecule. If the compound has a reactive group, then thecarrier molecule can alternatively be linked to the compound through thereactive group. The reactive group may contain both a reactivefunctional moiety and a linker, or only the reactive functional moiety.

A variety of carrier molecules are useful in the present invention.Exemplary carrier molecules include antigens, steroids, vitamins, drugs,haptens, metabolites, toxins, environmental pollutants, amino acids,peptides, proteins, nucleic acids, nucleic acid polymers, carbohydrates,lipids, and polymers. In another exemplary embodiment, at least onemember selected from R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, andR¹², is a carrier molecule or is attached to a carrier molecule. In oneaspect at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is a carriermolecule or is attached to a carrier molecule. In a further aspect, atleast one of R⁵, R⁶, R⁷ or R⁸ is or is a carrier molecule or is attachedto a carrier molecule.

In an exemplary embodiment, the carrier molecule comprises an aminoacid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof. In another exemplary embodiment, the carrier molecule isselected from a hapten, a nucleotide, an oligonucleotide, a nucleic acidpolymer, a protein, a peptide or a polysaccharide. In a preferredembodiment the carrier molecule is amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a tyramine, a synthetic polymer, a polymeric microparticle, abiological cell, cellular components, an ion chelating moiety, anenzymatic substrate or a virus. In another preferred embodiment, thecarrier molecule is an antibody or fragment thereof, an antigen, anavidin or streptavidin, a biotin, a dextran, an IgG binding protein, afluorescent protein, agarose, and a non-biological microparticle.

In an exemplary embodiment, the enzymatic substrate is selected from anamino acid, peptide, sugar, alcohol, alkanoic acid, 4-guanidinobenzoicacid, nucleic acid, lipid, sulfate, phosphate, —CH₂OCOalkyl andcombinations thereof. Thus, the enzyme substrates can be cleave byenzymes selected from the group consisting of peptidase, phosphatase,glycosidase, dealkylase, esterase, guanidinobenzotase, sulfatase,lipase, peroxidase, histone deacetylase, endoglycoceramidase,exonuclease, reductase and endonuclease.

In another exemplary embodiment, the carrier molecule is an amino acid(including those that are protected or are substituted by phosphates,carbohydrates, or C₁ to C₂₂ carboxylic acids), or a polymer of aminoacids such as a peptide or protein. In a related embodiment, the carriermolecule contains at least five amino acids, more preferably 5 to 36amino acids. Exemplary peptides include, but are not limited to,neuropeptides, cytokines, toxins, protease substrates, and proteinkinase substrates. Other exemplary peptides may function as organellelocalization peptides, that is, peptides that serve to target theconjugated compound for localization within a particular cellularsubstructure by cellular transport mechanisms. Preferred protein carriermolecules include enzymes, antibodies, lectins, glycoproteins, histones,albumins, lipoproteins, avidin, streptavidin, protein A, protein G,phycobiliproteins and other fluorescent proteins, hormones, toxins andgrowth factors. Typically, the protein carrier molecule is an antibody,an antibody fragment, avidin, streptavidin, a toxin, a lectin, or agrowth factor. Exemplary haptens include biotin, digoxigenin andfluorophores.

In another exemplary embodiment, the carrier molecule comprises anucleic acid base, nucleoside, nucleotide or a nucleic acid polymer,optionally containing an additional linker or spacer for attachment of afluorophore or other ligand, such as an alkynyl linkage (U.S. Pat. No.5,047,519), an aminoallyl linkage (U.S. Pat. No. 4,711,955) or otherlinkage. In another exemplary embodiment, the nucleotide carriermolecule is a nucleoside or a deoxynucleoside or a dideoxynucleoside.

Exemplary nucleic acid polymer carrier molecules are single- ormulti-stranded, natural or synthetic DNA or RNA oligonucleotides, orDNA/RNA hybrids, or incorporating an unusual linker such as morpholinederivatized phosphates (AntiVirals, Inc., Corvallis Oreg.), or peptidenucleic acids such as N-(2-aminoethyl)glycine units, where the nucleicacid contains fewer than 50 nucleotides, more typically fewer than 25nucleotides.

In another exemplary embodiment, the carrier molecule comprises acarbohydrate or polyol that is typically a polysaccharide, such asdextran, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, starch,agarose and cellulose, or is a polymer such as a poly(ethylene glycol).In a related embodiment, the polysaccharide carrier molecule includesdextran, agarose or FICOLL.

In another exemplary embodiment, the carrier molecule comprises a lipid(typically having 6-25 carbons), including glycolipids, phospholipids,and sphingolipids. Alternatively, the carrier molecule comprises a lipidvesicle, such as a liposome, or is a lipoprotein (see below). Somelipophilic substituents are useful for facilitating transport of theconjugated dye into cells or cellular organelles.

Alternatively, the carrier molecule is a cell, cellular systems,cellular fragment, or subcellular particles, including virus particles,bacterial particles, virus components, biological cells (such as animalcells, plant cells, bacteria, or yeast), or cellular components.Examples of cellular components that are useful as carrier moleculesinclude lysosomes, endosomes, cytoplasm, nuclei, histones, mitochondria,Golgi apparatus, endoplasmic reticulum and vacuoles.

In another exemplary embodiment, the carrier molecule non-covalentlyassociates with organic or inorganic materials. Exemplary embodiments ofthe carrier molecule that possess a lipophilic substituent can be usedto target lipid assemblies such as biological membranes or liposomes bynon-covalent incorporation of the dye compound within the membrane,e.g., for use as probes for membrane structure or for incorporation inliposomes, lipoproteins, films, plastics, lipophilic microspheres orsimilar materials.

In an exemplary embodiment, the carrier moelcule comprises a specificbinding pair member wherein the present compounds are conjugated to aspecific binding pair member and used to the formation of the boundpair. Alternatively, the presence of the labeled specific binding pairmember indicates the location of the complementary member of thatspecific binding pair; each specific binding pair member having an areaon the surface or in a cavity which specifically binds to, and iscomplementary with, a particular spatial and polar organization of theother. In this instance, the dye compounds of the present inventionfunction as a reporter molecule for the specific binding pair. Exemplarybinding pairs are set forth in Table 2.

TABLE 2 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor folate folate binding protein toxin toxin receptorcarbohydrate lectin or carbohydrate receptor peptide peptide receptorprotein protein receptor enzyme substrate enzyme DNA (RNA) cDNA (cRNA)†hormone hormone receptor ion chelator *IgG is an immunoglobulin †cDNAand cRNA are the complementary strands used for hybridization

Solid Supports

In an exemplary embodiment, the present compounds of the invention arecovalently bonded to a solid support. The solid support may be attachedto the compound either through the chelating moiety, reporter moiety, orthrough a reactive group, if present, or through a carrier molecule, ifpresent. Even if a reactive group and/or a carrier molecule are present,the solid support may be attached through the chelating moiety orreporter moiety. In another exemplary embodiment, at least one memberselected from R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹², isa solid support or is attached to a solid support. In one aspect, atleast one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is a solid support or isattached to a solid support. In a further aspect at least one of R⁵, R⁶,R⁷ or R⁸ is a solid support or is attached to a solid support.

A solid support suitable for use in the present invention is typicallysubstantially insoluble in liquid phases. Solid supports of the currentinvention are not limited to a specific type of support. Rather, a largenumber of supports are available and are known to one of ordinary skillin the art. Thus, useful solid supports include solid and semi-solidmatrixes, such as aerogels and hydrogels, resins, beads, biochips(including thin film coated biochips), microfluidic chip, a siliconchip, multi-well plates (also referred to as microtitre plates ormicroplates), membranes, conducting and nonconducting metals, glass(including microscope slides) and magnetic supports. More specificexamples of useful solid supports include silica gels, polymericmembranes, particles, derivatized plastic films, glass beads, cotton,plastic beads, alumina gels, polysaccharides such as Sepharose,poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar,cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin,mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride,polypropylene, polyethylene (including poly(ethylene glycol)), nylon,latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead,starch and the like.

In some embodiments, the solid support may include a solid supportreactive functional group, including, but not limited to, hydroxyl,carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea,carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide,sulfoxide, etc., for attaching the compounds of the invention. Usefulreactive groups are disclosed above and are equally applicable to thesolid support reactive functional groups herein.

A suitable solid phase support can be selected on the basis of desiredend use and suitability for various synthetic protocols. For example,where amide bond formation is desirable to attach the compounds of theinvention to the solid support, resins generally useful in peptidesynthesis may be employed, such as polystyrene (e.g., PAM-resin obtainedfrom Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE™ resin(obtained from Aminotech, Canada), polyamide resin (obtained fromPeninsula Laboratories), polystyrene resin grafted with polyethyleneglycol (TentaGel™, Rapp Polymere, Tubingen, Germany),polydimethyl-acrylamide resin (available from Milligen/Biosearch,California), or PEGA beads (obtained from Polymer Laboratories).

Preparation of Conjugates

Conjugates of components (carrier molecules or solid supports), e.g.,drugs, peptides, toxins, nucleotides, phospholipids and other organicmolecules are prepared by organic synthesis methods using the reactivedyes of the invention, are generally prepared by means well recognizedin the art (Haugland, MOLECULAR PROBES HANDBOOK, supra, (2002)).Preferably, conjugation to form a covalent bond consists of simplymixing the reactive compounds of the present invention in a suitablesolvent in which both the reactive compound and the substance to beconjugated are soluble. The reaction preferably proceeds spontaneouslywithout added reagents at room temperature or below. For those reactivecompounds that are photoactivated, conjugation is facilitated byillumination of the reaction mixture to activate the reactive compound.Chemical modification of water-insoluble substances, so that a desiredcompound-conjugate may be prepared, is preferably performed in anaprotic solvent such as dimethylformamide, dimethylsulfoxide, acetone,ethyl acetate, toluene, or chloroform. Similar modification ofwater-soluble materials is readily accomplished through the use of theinstant reactive compounds to make them more readily soluble in organicsolvents.

Preparation of peptide or protein conjugates typically comprises firstdissolving the protein to be conjugated in aqueous buffer at about. 1-10mg/mL at room temperature or below. Bicarbonate buffers (pH about 8.3)are especially suitable for reaction with succinimidyl esters, phosphatebuffers (pH about 7.2-8) for reaction with thiol-reactive functionalgroups and carbonate or borate buffers (pH about 9) for reaction withisothiocyanates and dichlorotriazines. The appropriate reactive compoundis then dissolved in a nonhydroxylic solvent (usually DMSO or DMF) in anamount sufficient to give a suitable degree of conjugation when added toa solution of the protein to be conjugated. The appropriate amount ofcompound for any protein or other component is convenientlypredetermined by experimentation in which variable amounts of thecompound are added to the protein, the conjugate is chromatographicallypurified to separate unconjugated compound and the compound-proteinconjugate is tested in its desired application.

Following addition of the reactive compound to the component solution,the mixture is incubated for a suitable period (typically about 1 hourat room temperature to several hours on ice), the excess compound isremoved by gel filtration, dialysis, HPLC, adsorption on an ion exchangeor hydrophobic polymer or other suitable means. The compound-conjugateis used in solution or lyophilized. In this way, suitable conjugates canbe prepared from antibodies, antibody fragments, avidins, lectins,enzymes, proteins A and G, cellular proteins, albumins, histones, growthfactors, hormones, and other proteins.

Conjugates of polymers, including biopolymers and other higher molecularweight polymers are typically prepared by means well recognized in theart (for example, Brinkley et al., Bioconjugate Chem., 3: 2 (1992)). Inthese embodiments, a single type of reactive site may be available, asis typical for polysaccharides) or multiple types of reactive sites(e.g. amines, thiols, alcohols, phenols) may be available, as is typicalfor proteins. Selectivity of labeling is best obtained by selection ofan appropriate reactive dye. For example, modification of thiols with athiol-selective reagent such as a haloacetamide or maleimide, ormodification of amines with an amine-reactive reagent such as anactivated ester, acyl azide, isothiocyanate or3,5-dichloro-2,4,6-triazine. Partial selectivity can also be obtained bycareful control of the reaction conditions.

When modifying polymers with the compounds, an excess of compound istypically used, relative to the expected degree of compoundsubstitution. Any residual, unreacted compound or a compound hydrolysisproduct is typically removed by dialysis, chromatography orprecipitation. Presence of residual, unconjugated dye can be detected bythin layer chromatography using a solvent that elutes the dye away fromits conjugate. In all cases it is usually preferred that the reagents bekept as concentrated as practical so as to obtain adequate rates ofconjugation.

In an exemplary embodiment, the conjugate of the invention is associatedwith an additional substance, that binds either to the fluorophore orthe conjugated substance (carrier molecule or solid support) throughnoncovalent interaction. In another exemplary embodiment, the additionalsubstance is an antibody, an enzyme, a hapten, a lectin, a receptor, anoligonucleotide, a nucleic acid, a liposome, or a polymer. Theadditional substance is optionally used to probe for the location of thedye-conjugate, for example, as a means of enhancing the signal of thedye-conjugate.

Synthesis

Typically the synthetic methodology used to prepare the compounds of theinvention involves preparation of an alkylene linker directly attachedto one of the phenyl ring carbons of the chelator moiety by acarbon-carbon bond. This can be achieved by a variety of syntheticoperations. Preparation of this linker is done in such as way so as toinclude a nucleophilic or pro-nucleophilic moiety attached to thelinker. The nucleophilic moiety on the linker is then covalentlyattached to a fluorophore by alkylation or acylation with a reactiveelectrophilic version of the fluorophore. The order of attachment can bereversed, i.e. the linker-fluorophore combination can be prepared first,followed by attachment to the chelator.

Method of Use

The metal ion chelating compounds of the invention are useful for anyapplication where it is desirable to complex a target metal ion.Selected compounds of the invention may be useful as ionophores, thatis, they facilitate the transport of selected target ions across cellmembranes. Where the present compound is bound to a conjugated substancethat is a polymeric matrix, such as a microparticle, or agarose, thecompounds are useful for depleting a sample solution of a selectedtarget ion, particularly where the polymeric matrix is used to pack achromatography column. Other compounds (those bound to a reportermoiety) are useful as fluorescent indicators for a selected target ion.

In order for a particular indicator of the present invention to beuseful for detection purposes, it must exhibit a detectable change inspectral properties upon complexation of the desired metal ion (targetion) in the chelating moiety. Preferably the change in spectralproperties is a change in fluorescence properties. More preferably, theinstant indicators display an intensity increase or decrease in emissionenergy upon the complexation of the desired target ion.

The present compounds are useful for binding target ions resulting in acomplex of the target ion and the present compounds. Therefore, anadditional aspect of the invention includes the compound of theinvention further comprising a metal ion that is associated and/orcomplexed within the chelate portion of the compound. The metal ion isoptionally Ca²⁺, Zn²⁺, Mg²⁺, Ga³⁺, Tb³⁺, La³⁺, Pb²⁺, Hg²⁺, Cd²⁺, Cu²⁺,Ni²⁺, Co²⁺, Fe²⁺, Mn²⁺, Ba²⁺, and Sr²⁺. Preferably the complex comprisesphysiological relevant cations such as Ca²⁺, Mg²⁺, Fe²⁺ and Zn²⁺.

Accordingly, a method for binding target metal ions in a samplecomprises the following steps:

-   -   a) contacting the sample with a fluorogenic chelate compound of        the present invention to form a contacted sample; and,    -   b) incubating the contacted sample for a sufficient amount of        time to allow the compound to bind the target metal ion whereby        the metal ion is bound.

When the present compounds are used as indicators a reporter moiety iscovalently attached to the chelate moiety via a present PET linker. Thesample is illuminated with an appropriate wavelength whereby the targetion is detected. In such an assay the target ion can also be quantitatedand monitored.

The specific indicator used in an assay or experiment is selected basedon the desired affinity for the target ion as determined by the expectedconcentration range in the sample, the desired spectral properties, andthe desired selectivity. Initially, the suitability of a material as anindicator of ion concentration is commonly tested by mixing a constantamount of the indicating reagent with a measured amount of the targetion under the expected experimental conditions.

Preferred indicators display a high selectivity, that is, they show asufficient rejection of non-target ions. The interference of anon-target ion is tested by a comparable titration of the indicator withthat ion. Although preferred target ions for most indicators of thepresent invention are Ca^(++, Na) ⁺ and K⁺, any ion that yields adetectable change in absorption wavelengths, emission wavelengths,fluorescence lifetimes or other measurable optical property over theconcentration range of interest is potentially measured using one of theindicators of this invention. Most preferred is calcium ions.

The indicator is generally prepared for use as a detection reagent bydissolving the indicator in solution at a concentration that is optimalfor detection of the indicator at the expected concentration of thetarget ion. Modifications that are designed to enhance permeability ofthe indicator through the membranes of living cells, such asacetoxymethyl esters and acetates, may require the indicator to bepredissolved in an organic solvent such as dimethylsulfoxide (DMSO)before addition to a cell suspension, where the indicators then readilyenter the cells. Intracellular enzymes cleave the esters to the morepolar acids and phenols that are then well retained inside the cells.For applications where permeability of cell-membranes is required, theindicators of the invention are typically substituted by only onefluorophore.

Therefore, a method for binding and detecting target ions in a live cellcomprises the following steps:

-   -   a) contacting a sample of live cells with a present compound        with the proviso that at least one of R¹³ or R¹⁴ is —CH₂OCOalkyl        or CH₃;    -   b) incubating the sample and the compound for sufficient time to        allow the compound to chelate the target metal ion; and,    -   c) illuminating the sample with an appropriate wavelength to        generate a detectable fluorescent signal that is modulated by        PET whereby the target ion is detected in a live cell.

A specific indicator of the present invention is useful for thedetection and/or quantification of a desired target ion, when thebinding of the target ion in the metal ion-binding moiety of theindicator results in a detectable change in spectral properties.Preferably, the change in spectral properties is a detectablefluorescence response.

The optical response of the indicating reagent is determined by changesin absorbance or fluorescence, preferably fluorescence. If absorbancemeasurements are used to determine ion concentrations, then it isusually optimal to adjust the optical density of the indicator in thesample over the range of analyte concentration to a value ofapproximately 0.02 to 2.5 (most preferably 0.1 to 1). For fluorescencemeasurements, the concentration of the indicator will depend mostly onthe sensitivity of the equipment used for its detection.

If the optical response of the indicator will be determined usingfluorescence measurements, samples are typically stained with indicatorconcentrations of 10⁻⁹ M to 10⁻² M. The most useful range of analyteconcentration is about one log unit above and below the dissociationconstant of the ion-indicator complex. This dissociation constant isdetermined by titration of the indicator with a known concentration ofthe target ion, usually over the range of virtually zero concentrationto approximately 100 millimolar of the target ion, depending on whichion is to be measured and which indicator is being used. Thedissociation constant may be affected by the presence of other ions,particularly ions that have similar ionic radii and charge. It may alsobe affected by other conditions such as ionic strength, pH, temperature,viscosity, presence of organic solvents and incorporation of the sensorin a membrane or polymeric matrix, or conjugation or binding of thesensor to a protein or other biological molecule. Any or all of theseeffects need to be taken into account when calibrating an indicator.

The indicator is combined with a sample in a way that will facilitatedetection of the target ion concentration in the sample. The sample isgenerally a representative cell population, fluid or liquid suspensionthat is known or suspected to contain the target ion. Representativesamples include intracellular fluids such as in blood cells, culturedcells, muscle tissue, neurons and the like; extracellular fluids inareas immediately outside of cells; in vesicles; in vascular tissue ofplants and animals; in biological fluids such as blood, saliva, andurine; in biological fermentation media; in environmental samples suchas water, soil, waste water and sea water; in industrial samples such aspharmaceuticals, foodstuffs and beverages; and in chemical reactors.Detection and quantitation of the target ion in a sample can helpcharacterize the identity of an unknown sample, or facilitate qualitycontrol of a sample of known origin.

In one embodiment of the invention, the sample contains cells, and theindicator is combined with the sample in such a way that the indicatoris present within the sample cells. By selection of the appropriatechelating moiety, fluorophore, and the substituents thereon, indicatorsare prepared that will selectively localize in desired organelles, andprovide measurements of the target ion in those organelles. Conjugatesof the indicators of the invention with organelle-targeting peptides areused to localize the indicator to the selected organelle, facilitatingmeasurement of target ion presence or concentration within the organelle(as described in U.S. Pat. No. 5,773,227). Alternatively, selection of alipophilic fluorophore, or a fluorophore having predominantly lipophilicsubstituents will result in localization in lipophilic environments inthe cell, such as cell membranes. Selection of cationic indicators willtypically result in localization of the indicator in mitochondria.

In another aspect of the invention, a composition of matter comprisesany of the compounds described above, and optionally includes a metalion. In one embodiment, the compounds of the invention, in any of theembodiments described above, are associated, either covalently ornoncovalently, with a surface such as a microfluidic chip, a siliconchip, a microscope slide, a microplate well, or another solid matrix,and is combined with the sample of interest as it flows over thesurface. The detectable optical response is therefore detected on thematrix surface itself, typically by use of an instrumental. Thisembodiment of the invention is particularly suited to high-throughputscreening using automated methods.

Quantification of target ion levels in samples is typically accomplishedusing the indicators of the present invention by methods known in theart. For example, the ratiometric measurement of ion concentrationprovides accurate measurement of ion concentrations by the treatment ofthe fluorescence data as the ratio of excitation or fluorescenceintensities at two wavelengths, rather than the absolute intensity at asingle wavelength. Using the ratio method, a number of variables thatmay perturb the ion concentration measurements are eliminated. Inparticular, ion-dependent factors that affect the signal intensity, suchas nonuniform intracellular dye concentrations, probe leakage, dyebleaching and cell thickness, are canceled in the ratio measurements,since these parameters have a similar effect on intensities at bothwavelengths. While the ratio method can be used to determineconcentrations using observation of either the excitation spectra of theindicator, the emission spectra of the indicator, or both, in the caseof the indicators of the present invention, the shift in excitationenergy upon binding metal ions makes observation of the excitationspectrum a more useful technique. In either case, to achieve maximalutility, the indicator must be calibrated (to compensate for variance inthe dissociation constant of the indicator due to ionic strength,viscosity, or other conditions within the sample). To calibrate theindicator, ionophores such as A-23187, gramicidin, valinomycin, orionomycin are used. Non-ratiometric analysis can also be accomplished bycalibration with a second fluorescent dye present in the sample.

The optical response of the indicator to the ion can be detected byvarious means that include measuring absorbance or fluorescence changeswith an instrument, visually, or by use of a fluorescence sensingdevice. Several examples of fluorescence sensing devices are known, suchas fluorometers, fluorescence microscopes, laser scanners, flowcytometers, and microfluidic devices, as well as by cameras and otherimaging equipment. These measurements may be made remotely byincorporation of the fluorescent ion sensor as part of a fiber opticprobe. The indicator is covalently attached to the fiber optic probematerial, typically glass or functionalized glass (e.g., aminopropylglass) or the indicator is attached to the fiber optic probe via anintermediate polymer, such as polyacrylamide. The indicator solution isalternatively incorporated non-covalently within a fiber optic probe, aslong as there is a means whereby the target ion can come into contactwith the indicator solution.

C. Kits of the Invention

Due to the advantageous properties and the simplicity of use of theinstant metal ion-binding compounds, they are particularly useful in theformulation of a kit for the complexation, detection, quantification ormonitoring of selected target ions, comprising one or more compounds orcompositions of the invention in any of the embodiments described above(optionally in a stock solution), instructions for the use of the crownether compound to complex or detect a desired target ion, and optionallycomprising additional components. In one aspect, the compounds of theinvention are associated with a surface, such as a chip, microplatewell, or other solid matrix, and the sample of interest flows over thesurface. The detectable optical response is therefore detected on thematrix surface itself.

Therefore a kit of the present invention for binding a target metal ionin a sample comprises a present compound and instructions for usethereof. The kit may further comprise one or more components selectedfrom the group consisting of a calibration standard of a metal ion, anionophore, a fluorescent standard, an aqueous buffer solution and anorganic solvent.

The additional kit components may be selected from, without limitation,calibration standards of a target ion, ionophores, fluorescencestandards, aqueous buffers, and organic solvents. The additional kitcomponents are present as pure compositions, or as aqueous solutionsthat incorporate one or more additional kit components. Any or all ofthe kit components optionally further comprise buffers.

The examples below are given so as to illustrate the practice of thisinvention. They are not intended to limit or define the entire scope ofthis invention.

EXAMPLES Example 1 Synthesis of 5-(Aminomethyl) BAPTA

To a stirred suspension of 5-formyl BAPTA 1 (5.60 g, 10 mmol) in EtOH(100 mL) was added a solution of hydroxylamine hydrochloride (1.40 g, 20mmol) in H₂O (5 mL) followed by 3N NaOAc (5 mL, 15 mmol). The mixturewas stirred at 60° C. for 4 h, cooled to room temperature andevaporated. Water (200 mL) was added to the residue, the productfiltered and washed with water (10×50 mL), dried in air, then in vacuoto give oxime 2, 5.37 g (91%) as an off-white solid. The product 2 doesnot require purification to use in the next step.

To a stirred solution of oxime 2 (4.71 g, 8 mmol) in acetic acid (80mL), powdered Zn (2.62 g, 40 mmol) was added in one portion. The mixturewas stirred for 6 h, diluted with CHCl₃ (300 mL) and filtered frominorganic material. The filtrate was evaporated and the residue loadedonto a SiO₂ column (3×30 cm bed, made in 5% MeOH and 1% AcOH in CHCl₃).The product was eluted with a gradient of 5-20% MeOH in CHCl₃ with 1%AcOH to give the amine 3, 2.72 g (59%) as an off-white solidified oil.

Example 2 Synthesis of 5-(Dansylaminomethyl) BAPTA derivatives

To a solution of amine 3 (160 mg, 0.28 mmol) in pyridine (3 mL), drypowdered dansyl chloride (98 mg, 0.36 mmol) was added in three portionswithin 5 min. The mixture was stirred for 2 h, then evaporated. Thesolid residue was dissolved in CHCl₃ (100 mL), washed with H₂O (100 mL),1% AcOH (2×100 mL), and sat. NaCl (100 mL). Chloroform was evaporatedand the residue was purified by preparative TLC on silica gel using 3%MeOH in CHCl₃ as eluant to give 5-(dansylaminomethyl) BAPTA, tetramethylester 4, 60 mg (27%) as a white powder.

A mixture of tetramethyl ester 4 (60 mg, 0.074 mmol), MeOH (1 mL),dioxane (1 mL), H₂O (1 mL), and 1N KOH (0.75 mL, 0.75 mmol.) was stirredfor 16 h, then 0.2 N HCl added to achieve pH 9.5, and the mixture wasevaporated. The residue was purified by column chromatography onSephadex LH-20 (3.5×50 cm bed, made in H₂O) using H₂O as eluant to give5-(dansylaminomethyl) BAPTA, tetrapotassium salt 5, 46 mg (69%) as anoff-white solid after lyophilization.

A mixture of compound 5 (9 mg, 0.01 mmol), bromomethyl acetate (10 μL,0.1 mmol), and N,N-diisopropylethylamine (DIEA, 35 μL, 0.2 mmol) in DMF(0.3 mL) was stirred for 1 h, and evaporated. The residue was purifiedby preparative TLC on silica gel using 2% MeOH in CHCl₃ as eluant togive 5-(dansylaminomethyl) BAPTA, tetra(acetoxymethyl) ester 6, 5 mg(48%) as an off-white solid.

Example 3 Synthesis of 5-(N-(5′-fluoresceinyl)aminomethyl) derivatives

To a stirred solution of amine 3 (115 mg, 0.2 mmol) and DIEA (0.17 mL,1.0 mmol) in CH₂Cl₂ (5 mL) was added a solution of acyl chloride 8,prepared from the acid 7 (149 mg, 0.3 mmol) and oxalyl chloride (0.1 mL,1.2 mmol). The mixture was stirred 16 h, then diluted with CHCl₃ (100mL), and washed with 1% AcOH (2×20 mL), sat. NaCl, filtered andevaporated. The residue was purified by preparative TLC on silica gelusing 5% MeOH in CHCl₃ as eluant to give tetramethyl ester 9, 71 mg(34%) as an off-white solid.

A mixture of tetramethyl ester 9 (50 mg, 0.047 mmol), MeOH (2 mL),dioxane (2 mL), and 1N KOH (0.5 mL, 0.5 mmol.) was stirred for 16 h,then 0.2 N HCl added to pH 9.0, and the mixture was evaporated. Theresidue was purified by column chromatography on Sephadex LH-20 (6×70 cmbed, made in H₂O) using H₂O as eluant to give hexapotassium salt 10, 39mg (72%) as a yellow solid (after lyophilization).

Example 4 Synthesis of 5-(1,8-naphthaleneimidomethyl) BAPTA derivatives

A mixture of amine 3 (1.46 g, 2.5 mmol) and 4-amino-1,8-naphthalicanhydride 11 (0.64 g, 3.0 mmol) in DMF (10 mL) was stirred at 60° C. for20 h, cooled to room temperature and diluted with CHCl₃ (400 mL). Themixture was washed with sat. NaHCO₃ (5×500 mL), H₂O (500 mL), sat. NaCl(500 mL), filtered and evaporated. The residue was purified bychromatography on a silica gel column (6×50 cm bed, made with 45% EtOAcin hexanes) using an EtOAC gradient (45-70%) in hexanes to give compound12, 0.26 g (14%) as a yellow solid.

A solution of tetramethyl ester 12 (210 mg, 0.27 mmol) in MeOH (30 mL)and 1N KOH (2.7 mL, 1.0 mmol.) was stirred for 16 h, then 0.2 N HCl wasadded to pH 8.5 and the mixture was evaporated. The residue was purifiedby column chromatography on Sephadex LH-20 (6×70 cm bed, made in H₂O)using H₂O as eluant to give tetrapotassium salt 13, 210 mg (89%) as ayellow solid after lyophilization.

A solution of tetramethyl ester 12 (77 mg, 0.1 mmol) in MeOH (1 mL),dioxane (2 mL), and 1N KOH (1.0 mL, 1.0 mmol.) was stirred for 16 h,then evaporated. The residue was dissolved in H₂O (10 mL) and 0.2 N HClwas added to pH 3.5. The resulting precipitate was filtered, washed withcold water (3 mL), and dried in vacuo to give compound 1458 mg (80%) asan orange solid. Tetraacid 14 was used in the next step withoutpurification.

A mixture of compound 14 (44 mg, 0.05 mmol), bromomethyl acetate (50 L,0.5 mmol) and N,N-diisopropylethylamine (175 μL, 1.0 mmol) in DMF (2 mL)was stirred for 16 h and evaporated. The residue was purified bypreparative TLC on silica gel using 50% EtOAc in hexanes as eluant togive tetra(acetoxymethyl) ester 15, 26 mg (52%) as an orange solid.

Example 5 Synthesis of 5-(N-(resorufinyl)aminomethyl) BAPTA derivatives

To a stirred solution of amine 3 (115 mg, 0.2 mmol) and DIEA (0.17 mL,1.0 mmol) in DMF (2 mL) was added a solution of(N-hydroxysuccinimidylcarboxy)resorufin 16 (50 mg, 0.14 mmol) in DMF (2mL). The mixture was stirred 16 h, then diluted with CHCl₃ (100 mL), andwashed with 1% AcOH (3×50 mL), sat. NaCl (100 mL), filtered andevaporated. The residue was purified by preparative TLC on silica gelusing 5% MeOH and 1% AcOH in CHCl₃ as eluant to give tetramethyl ester17, 80 mg (70%) as a dark red solid.

A mixture of tetramethyl ester 17 (80 mg, 0.1 mmol) and LiI (1.340 g, 10mmol.) in anhydrous acetonitrile (10 mL) was refluxed for 16 h. Theresulted dark solution was cooled to room temperature and the crudeproduct was collected by filtration and washed with acetonitrile (5×5mL). It was purified by column chromatography on Sephadex LH-20 (2.6×90cm bed, made in H₂O) using H₂O as eluant to give tetralithiium salt 18,15 mg (19%) as a dark purple solid (after lyophilization).

Example 6 Synthesis of 5-(N-(azarhodol)aminomethyl) BAPTA derivatives

To a stirred solution of amine 3 (58 mg, 0.1 mmol) and DIEA (0.09 mL,0.5 mmol) in DMF (3 mL) was added a solution of(N-hydroxysuccinimidylcarboxy)azarhodol 19 (40 mg, 0.1 mmol). Themixture was stirred 6 h, then diluted with CHCl₃ (100 mL), and washedwith 1% AcOH (3×100 mL), water (2×100 mL), sat. NaCl (100 mL), filteredand evaporated. The residue was purified by preparative TLC on silicagel using 5% MeOH and 2% AcOH in CHCl₃ as eluant to give tetramethylester 20, 45 mg (54%) as a dark blue solid.

A mixture of tetramethyl ester 20 (20 mg, 0.024 mmol) and LiI (319 mg,2.4 mmol.) in anhydrous acetonitrile (5 mL) was refluxed for 16 h. Theresulted dark solution was cooled to room temperature and the crudeproduct was collected by filtration and washed with acetonitrile (5×5mL). It was purified by column chromatography on Sephadex LH-20 (1.7×70cm bed, made in H₂O) using H₂O as eluant to give tetralithiium salt 21,13 mg (67%) as a dark purple solid (after lyophilization).

Example 7 Calcium Binding of Certain Calcium Binding Compounds

In all cases, samples were dissolved in nanopure(np)-H₂O atconcentrations of approximately 1 mg/mL, except Compound 18 which wasdissolved in 1:1 DMSO:npH2O, Non-quantitative aliquots of thesesolutions were added to approximately 3 mL of 39 μM Ca++ buffer, 100 mMKCl, 30 mM MOPS (pH7.2) (Compounds 5, 10, 13), Zero Ca++ buffer, 100 mMKCl, 30 mM MOPS (pH7.2) (Compound 18), or npH2O (Compound 21).Absorption spectra were obtained using a Perkin Elmer UV/VisSpectrometer (Lambda 35 or 40). Fluorescence samples were prepared bypipetting aliquots of respective stock solutions to a series ofdisposable cuvettes containing 2 mL of Ca++ buffers from CalciumCalibration Buffer Kit #2 (Invitrogen cat. #3009) (0-39 μM). The finalconcentration of compound ranged from approximately 0.1-4 μM. Sampleswere excited at their wavelength of maximum absorption and spectra wereobtained using a Perkin Elmer Luminescence Spectrometer (LS50B, LS55).Emission intensities were recorded at the respective maxima and Kdvalues calculated. See Table 2.

TABLE 2 Binding properties of the certain calcium binding compoundsK_(d) (Ca), Compound Fluorophore λ_(ex) (nm) λ_(em) (nm nM ΔF =Fb/Fo^(i) 5 Dansyl 331 549 81 3 10 Oregon 494 522 86 2 Green 13 Lucifer432 550 161 12 Yellow 18 Resorufin 550 589 271 5 21 AzaRhodol 612 641219 11

All patents and patent applications referred to within this document areincorporated by reference to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference.

The reagents employed in the examples are commercially available or canbe prepared using commercially available instrumentation, methods, orreagents known in the art. The foregoing examples illustrate variousaspects of the invention and practice of the methods of the invention.The examples are not intended to provide an exhaustive description ofthe many different embodiments of the invention. Thus, although theforgoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, thoseof ordinary skill in the art will realize readily that many changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims.

1. A compound for the detection of metal ions wherein a detectableresponse is modulated by photoinduced electron transfer (PET), whereinthe compound comprises a metal chelating moiety and a fluorophore or afluorescent protein that is covalently bonded to the metal chelatingmoiety by a linker —(CR2)nNR′— or —(CR2)n- wherein R and R′ areindependently hydrogen, alkyl, or substituted alkyl; and n is 1-10. 2.(canceled)
 3. A compound of the formula:

wherein R¹⁵ is hydrogen or C₁-C₆ alkyl; R¹⁶ is hydrogen or C₁-C₆ alkyl;R¹⁷ is hydrogen or C₁-C₆ alkyl; R¹⁸ is hydrogen or C₁-C₆ alkyl; and R¹³is hydrogen, C₁-C₆ alkyl, —CH₂OCOCH₃ or a salt ion; R¹⁴ is hydrogen,C₁-C₆ alkyl, —CH₂OCOCH₃ or a salt ion; R¹ is hydrogen, halogen, C₁ toC₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆ alkoxy (—OCH₂),alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR¹⁹R²⁰),aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl,reactive group, carrier molecule, solid support, reporter molecule,—(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore; R²is hydrogen, halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl(—OH), C₂-C₆ alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl,heteroaryl, amino (—NR¹⁹R²⁰), aldehyde, carboxyl, azido, nitro, nitroso,cyano, thioether, sulfonyl, reactive group, carrier molecule, solidsupport, reporter molecule, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophoreor —(CR₂)_(n)-fluorophore; R³ is hydrogen, halogen, C₁ to C₁₀ alkyl(CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆ alkoxy (—OCH₂), alicyclic,heteroalicyclic, aryl, heteroaryl, amino (—NR¹⁹R²⁰), aldehyde, carboxyl,azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group,carrier molecule, solid support, reporter molecule, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore; R⁴ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl, heteroaryl, amino(—NR¹⁹R²⁰), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether,sulfonyl, reactive group, carrier molecule, solid support, reportermolecule, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore; R⁵ is hydrogen, halogen, C₁ to C₁₀ alkyl (CH₂),methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆ alkoxy (—OCH₂), alicyclic,heteroalicyclic, aryl, heteroaryl, amino (—NR¹⁹R²⁰), aldehyde, carboxyl,azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group,carrier molecule, solid support, reporter molecule, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore; R⁶ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl, heteroaryl, amino(—NR¹⁹R²⁰), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether,sulfonyl, reactive group, carrier molecule, solid support, reportermolecule, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore; R⁷ is hydrogen, halogen, C₁ to C₁₀ alkyl (CH₂),methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆ alkoxy (—OCH₂), alicyclic,heteroalicyclic, aryl, heteroaryl, amino (—NR¹⁹R²⁰), aldehyde, carboxyl,azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group,carrier molecule, solid support, reporter molecule, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore; R⁸ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl, heteroaryl, amino(—NR¹⁹R²⁰), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether,sulfonyl, reactive group, carrier molecule, solid support, reportermolecule, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore; or a member independently selected from R¹ incombination with R²; R² in combination with R³; R³ in combination withR⁴; R⁵ in combination with R⁶; R⁶ in combination with R⁷; and R⁷ incombination with R⁸ together with the atoms to which they are joined,form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-memberedheteroaryl; wherein R is hydrogen, alkyl or substituted alkyl; R′ ishydrogen, alkyl, or substituted alkyl; R″ is hydrogen, alkyl,substituted alkyl, fluorophore, carrier molecule, solid support orreactive group; and n is 1-10; and, wherein R¹⁹ is hydrogen, C₁-C₆alkyl, substituted alkyl, C₁-C₆ carboxyalkyl (—(CH₂)₁₋₆COOR¹³), analpha-acyloxyalkyl, a biologically compatible salt, aryl, substitutedaryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substitutedheteroaryl; R²⁰ is hydrogen, C₁-C₆ alkyl, substituted alkyl, C₁-C₆carboxyalkyl (—(CH₂)₁₋₆COOR¹³), an alpha-acyloxyalkyl, a biologicallycompatible salt, aryl, substituted aryl, aryl alkyl, substituted arylalkyl, heteroaryl, and substituted heteroaryl; R⁹ is hydrogen, areactive group, a carrier molecule, a solid support, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore and C₁-C₆ alkyl; R¹⁰ is hydrogen, a reactivegroup, a carrier molecule, a solid support, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore and C₁-C₆ alkyl; R¹¹ is hydrogen, a reactivegroup, a carrier molecule, a solid support, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore and C₁-C₆ alkyl; R¹² is hydrogen, a reactivegroup, a carrier molecule, a solid support, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore and C₁-C₆ alkyl; or a member selected from R⁹in combination with R¹⁰; or R¹¹ in combination with R¹² together withthe atoms to which they are joined, form a ring which is a 5-, or6-membered alicyclic ring, a substituted 5-, or 6-membered alicyclicring, a 5-, or 6-membered heterocyclic ring, or a substituted 5-, or6-membered heterocyclic ring; p is 0, 1, 2 or 3; and wherein at leastone of R¹-R¹² is —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore with the proviso that when at least one of R¹-R¹²is —(CR₂)_(n)-fluorophore that the fluorophore comprise a nitrogen atomthat is covalently bonded to —(CR₂)_(n)—.
 4. The compound according toclaim 3, wherein R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are each hydrogen.
 5. Thecompound according to claim 3, wherein R¹³ and R¹⁴ are independentlyhydrogen or a salt ion.
 6. The compound according to claim 3, whereinR¹³ and R¹⁴ are independently —CH₂OCOCH₃.
 7. The compound according toclaim 3, wherein exactly one of R¹-R⁸ is —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore.
 8. The compound according to claim 3, whereinexactly one of R², R³, R⁶ or R⁷ is —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore.
 9. The compound according to claim 3, wherein Rand R′ are each hydrogen.
 10. The compound according to claim 3, whereinthe fluorophore is dansyl, xanthene, cyanine, borapolyazaindacene,pyrene, naphthalene, coumarin, oxazine or derivatives thereof.
 11. Thecompound according to claim 10, wherein the xanthene is fluorescein orderivatives thereof, rhodamine or derivatives thereof, rhodol orderivatives thereof, or rosamine or derivatives thereof.
 12. Thecompound according to claim 11, wherein the xanthene is substituted byat least one fluorine.
 13. The compound according to claim 3, whereinthe reactive group, solid support and carrier molecule comprise a linkerthat is a single covalent bond, or a covalent linkage that is linear orbranched, cyclic or heterocyclic, saturated or unsaturated, having 1-20nonhydrogen atoms selected from the group consisting of C, N, P, O andS; and are composed of any combination of ether, thioether, amine,ester, carboxamide, sulfonamide, hydrazide bonds and aromatic orheteroaromatic bonds.
 14. The compound according to claim 3, wherein thereactive group is selected from the group consisting of carboxylic acid,succinimidyl ester of a carboxylic acid, hydrazide, amine and amaleimide.
 15. The compound according to claim 3, wherein the carriermolecule is selected from the group consisting of an amino acid, apeptide, a protein, a polysaccharide, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, ahormone, a lipid, a lipid assembly, a synthetic polymer, a polymericmicroparticle, a biological cell or a virus.
 16. The compound accordingto claim 3, wherein the solid support is selected from the groupconsisting of a microfluidic chip, a silicon chip, a microscope slide, amicroplate well, silica gels, polymeric membranes, particles,derivatized plastic films, glass beads, cotton, plastic beads, aluminagels, polysaccharides, polyvinylchloride, polypropylene, polyethylene,nylon, latex bead, magnetic bead, paramagnetic bead, andsuperparamagnetic bead.
 17. The compound according to claim 3, havingthe formula:

wherein R¹³ is hydrogen, C₁-C₆ alkyl, —CH₂OCOCH₃ or a salt ion; R¹⁴ ishydrogen, C₁-C₆ alkyl, —CH₂OCOCH₃ or a salt ion; R¹ is hydrogen,halogen, or C₁ to C₁₀ alkyl (CH₂), carboxyl, sulfonyl; R² is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), carboxyl, sulfonyl; R⁴ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), carboxyl, sulfonyl; R⁵ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), carboxyl, sulfonyl; R⁶ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), carboxyl, sulfonyl; R⁷ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), carboxyl, sulfonyl; R⁸ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), carboxyl, sulfonyl; R′ is hydrogen,alkyl, or substituted alkyl; R″ is a fluorophore, carrier and n is 1-10.18. The compound according to claim 3, wherein the compound is selectedfrom the group consisting of Compound 5, 6, 9, 10, 13, 14 and
 15. 19. Acomposition comprising: a) a compound having the formula:

wherein R¹⁵ is hydrogen or C₁-C₆ alkyl; R¹⁶ is hydrogen or C₁-C₆ alkyl;R¹⁷ is hydrogen or C₁-C₆ alkyl; R¹⁸ is hydrogen or C₁-C₆ alkyl; and R¹³is hydrogen, C₁-C₆ alkyl, —CH₂OCOCH₃ or a salt ion; R¹⁴ is hydrogen,C₁-C₆ alkyl, —CH₂OCOCH₃ or a salt ion; R¹ is hydrogen, halogen, C₁ toC₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆ alkoxy (—OCH₂),alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR¹⁹R²⁰),aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl,reactive group, carrier molecule, solid support, reporter molecule,—(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore; R²is hydrogen, halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl(—OH), C₂-C₆ alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl,heteroaryl, amino (—NR¹⁹R²⁰), aldehyde, carboxyl, azido, nitro, nitroso,cyano, thioether, sulfonyl, reactive group, carrier molecule, solidsupport, reporter molecule, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophoreor —(CR₂)_(n)-fluorophore; R³ is hydrogen, halogen, C₁ to C₁₀ alkyl(CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆ alkoxy (—OCH₂), alicyclic,heteroalicyclic, aryl, heteroaryl, amino (—NR¹⁹R²⁰), aldehyde, carboxyl,azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group,carrier molecule, solid support, reporter molecule, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore; R⁴ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl, heteroaryl, amino(—NR¹⁹R²⁰), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether,sulfonyl, reactive group, carrier molecule, solid support, reportermolecule, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore; R⁵ is hydrogen, halogen, C₁ to C₁₀ alkyl (CH₂),methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆ alkoxy (—OCH₂), alicyclic,heteroalicyclic, aryl, heteroaryl, amino (—NR¹⁹R²⁰), aldehyde, carboxyl,azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group,carrier molecule, solid support, reporter molecule, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore; R⁶ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl, heteroaryl, amino(—NR¹⁹R²⁰), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether,sulfonyl, reactive group, carrier molecule, solid support, reportermolecule, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore; R⁷ is hydrogen, halogen, C₁ to C₁₀ alkyl (CH₂),methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆ alkoxy (—OCH₂), alicyclic,heteroalicyclic, aryl, heteroaryl, amino (—NR¹⁹R²⁰), aldehyde, carboxyl,azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group,carrier molecule, solid support, reporter molecule, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore; R⁸ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl, heteroaryl, amino(—NR¹⁹R²⁰), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether,sulfonyl, reactive group, carrier molecule, solid support, reportermolecule, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore; or a member independently selected from R¹ incombination with R²; R² in combination with R³; R³ in combination withR⁴; R⁵ in combination with R⁶; R⁶ in combination with R⁷; and R⁷ incombination with R⁸ together with the atoms to which they are joined,form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-memberedheteroaryl; wherein R is hydrogen, alkyl or substituted alkyl; R′ ishydrogen, alkyl, or substituted alkyl; R″ is hydrogen, alkyl,substituted alkyl, fluorophore, carrier molecule, solid support orreactive group; and n is 1-10; and, wherein R¹⁹ is hydrogen, C₁-C₆alkyl, substituted alkyl, C₁-C₆ carboxyalkyl (—(CH₂)₁₋₆COOR¹³), analpha-acyloxyalkyl, a biologically compatible salt, aryl, substitutedaryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substitutedheteroaryl; R²⁰ is hydrogen, C₁-C₆ alkyl, substituted alkyl, C₁-C₆carboxyalkyl (—(CH₂)₁₋₆COOR¹³), an alpha-acyloxyalkyl, a biologicallycompatible salt, aryl, substituted aryl, aryl alkyl, substituted arylalkyl, heteroaryl, and substituted heteroaryl; R⁹ is hydrogen, areactive group, a carrier molecule, a solid support, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore and C₁-C₆ alkyl; R¹⁰ is hydrogen, a reactivegroup, a carrier molecule, a solid support, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore and C₁-C₆ alkyl; R¹¹ is hydrogen, a reactivegroup, a carrier molecule, a solid support, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore and C₁-C₆ alkyl; R¹² is hydrogen, a reactivegroup, a carrier molecule, a solid support, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore and C₁-C₆ alkyl; or a member selected from R⁹in combination with R¹⁰; or R¹¹ in combination with R¹² together withthe atoms to which they are joined, form a ring which is a 5-, or6-membered alicyclic ring, a substituted 5-, or 6-membered alicyclicring, a 5-, or 6-membered heterocyclic ring, or a substituted 5-, or6-membered heterocyclic ring; p is 0, 1, 2 or 3; and wherein at leastone of R¹-R¹² is —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore with the proviso that when at least one of R¹-R¹²is —(CR₂)_(n)-fluorophore that the fluorophore comprise a nitrogen atomthat is covalently bonded to —(CR₂)_(n)—; and b) a metal ion that iscapable of being chelated by the compound.
 20. A composition accordingto claim 19, wherein the metal ion is selected from the group consistingof Ca²⁺, Zn²⁺, Mg²⁺, Ga³⁺, Tb³⁺, La³⁺, Pb²⁺, Hg²⁺, Cd²⁺, Cu²⁺, Ni²⁺,Co²⁺, Fe²⁺, Mn²⁺, Ba²⁺, and Sr²⁺. 21.-32. (canceled)
 33. The compoundaccording to claim 1, wherein the compound has a structure of theformula:

wherein R¹⁵ is hydrogen or C₁-C₆ alkyl; R¹⁶ is hydrogen or C₁-C₆ alkyl;R¹⁷ is hydrogen or C₁-C₆ alkyl; R¹⁸ is hydrogen or C₁-C₆ alkyl; and R¹³is hydrogen, C₁-C₆ alkyl, —CH₂OCOCH₃ or a salt ion; R¹⁴ is hydrogen,C₁-C₆ alkyl, —CH₂OCOCH₃ or a salt ion; R¹ is hydrogen, halogen, C₁ toC₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆ alkoxy (—OCH₂),alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR¹⁹R²⁰),aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl,reactive group, carrier molecule, solid support, reporter molecule,—(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore; R²is hydrogen, halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl(—OH), C₂-C₆ alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl,heteroaryl, amino (—NR¹⁹R²⁰), aldehyde, carboxyl, azido, nitro, nitroso,cyano, thioether, sulfonyl, reactive group, carrier molecule, solidsupport, reporter molecule, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophoreor —(CR₂)_(n)-fluorophore; R³ is hydrogen, halogen, C₁ to C₁₀ alkyl(CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆ alkoxy (—OCH₂), alicyclic,heteroalicyclic, aryl, heteroaryl, amino (—NR¹⁹R²⁰), aldehyde, carboxyl,azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group,carrier molecule, solid support, reporter molecule, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore; R⁴ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl, heteroaryl, amino(—NR¹⁹R²⁰), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether,sulfonyl, reactive group, carrier molecule, solid support, reportermolecule, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore; R⁵ is hydrogen, halogen, C₁ to C₁₀ alkyl (CH₂),methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆ alkoxy (—OCH₂), alicyclic,heteroalicyclic, aryl, heteroaryl, amino (—NR¹⁹R²⁰), aldehyde, carboxyl,azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group,carrier molecule, solid support, reporter molecule, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore; R⁶ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl, heteroaryl, amino(—NR¹⁹R²⁰), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether,sulfonyl, reactive group, carrier molecule, solid support, reportermolecule, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore; R⁷ is hydrogen, halogen, C₁ to C₁₀ alkyl (CH₂),methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆ alkoxy (—OCH₂), alicyclic,heteroalicyclic, aryl, heteroaryl, amino (—NR¹⁹R²⁰), aldehyde, carboxyl,azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group,carrier molecule, solid support, reporter molecule, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore or —(CR₂)_(n)-fluorophore; R⁸ is hydrogen,halogen, C₁ to C₁₀ alkyl (CH₂), methoxy (—OCH₃), hydroxyl (—OH), C₂-C₆alkoxy (—OCH₂), alicyclic, heteroalicyclic, aryl, heteroaryl, amino(—NR¹⁹R²⁰), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether,sulfonyl, reactive group, carrier molecule, solid support, reportermolecule, —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore; or a member independently selected from R¹ incombination with R²; R² in combination with R³; R³ in combination withR⁴; R⁵ in combination with R⁶; R⁶ in combination with R⁷; and R⁷ incombination with R⁸ together with the atoms to which they are joined,form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-memberedheteroaryl; wherein R is hydrogen, alkyl or substituted alkyl; R′ ishydrogen, alkyl, or substituted alkyl; R″ is hydrogen, alkyl,substituted alkyl, fluorophore, carrier molecule, solid support orreactive group; and n is 1-10; and, wherein R¹⁹ is hydrogen, C₁-C₆alkyl, substituted alkyl, C₁-C₆ carboxyalkyl (—(CH₂)₁₋₆COOR¹³), analpha-acyloxyalkyl, a biologically compatible salt, aryl, substitutedaryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substitutedheteroaryl; R²⁰ is hydrogen, C₁-C₆ alkyl, substituted alkyl, C₁-C₆carboxyalkyl (—(CH₂)₁₋₆COOR¹³), an alpha-acyloxyalkyl, a biologicallycompatible salt, aryl, substituted aryl, aryl alkyl, substituted arylalkyl, heteroaryl, and substituted heteroaryl; R⁹ is hydrogen, areactive group, a carrier molecule, a solid support, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore and C₁-C₆ alkyl; R¹⁰ is hydrogen, a reactivegroup, a carrier molecule, a solid support, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore and C₁-C₆ alkyl; R¹¹ is hydrogen, a reactivegroup, a carrier molecule, a solid support, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore and C₁-C₆ alkyl; R¹² is hydrogen, a reactivegroup, a carrier molecule, a solid support, —(CR₂)_(n)NR′R″,—(CR₂)_(n)NR′-fluorophore and C₁-C₆ alkyl; or a member selected from R⁹in combination with R¹⁰; or R¹¹ in combination with R¹² together withthe atoms to which they are joined, form a ring which is a 5-, or6-membered alicyclic ring, a substituted 5-, or 6-membered alicyclicring, a 5-, or 6-membered heterocyclic ring, or a substituted 5-, or6-membered heterocyclic ring; p is 0, 1, 2 or 3; and wherein at leastone of R¹-R¹² is —(CR₂)_(n)NR′R″, —(CR₂)_(n)NR′-fluorophore or—(CR₂)_(n)-fluorophore with the proviso that when at least one of R¹-R¹²is —(CR₂)_(n)-fluorophore then the fluorophore comprises a nitrogen atomcovalently bonded to —(CR₂)_(n)—.
 34. The compound according to claim 1,wherein the metal ion is Ca²⁺, Zn²⁺, Mg²⁺, Ga³⁺, Tb³⁺, La³⁺, Pb²⁺, Hg²⁺,Cd²⁺, Cu²⁺, Ni²⁺, Co²⁺, Fe²⁺, Mn²⁺, Ba²⁺, or Sr²⁺.
 35. The compoundaccording to claim 1, wherein the detectable response produced by thecompound is a fluorescent response exhibiting a Stokes shift of greaterthan 50 nm.